Compositions and methods for the treatment of immune related diseases

ABSTRACT

Compositions and methods for improving detection sensitivity in nucleic acid microarray analysis are disclosed, including methods of purifying nucleic acids, methods of synthesizing fluorescent DNA probes, methods of hybridization, and methods of activating a substrate for target molecule attachment are disclosed.

FIELD OF THE INVENTION

This invention relates to compositions and methods for improved analysisof gene expression, genetic polymorphism or gene mutation using nucleicacid microarrays for genetic research and diagnostic applications.

BACKGROUND

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differential gene expression in diseasedtissue relative to normal tissue of the same type, for example. Usingnucleic acid microarrays, test and control mRNA samples from test andcontrol tissue samples are reverse transcribed and labeled to generatecDNA probes. The probes are then hybridized to an array of nucleic acidsimmobilized on a solid support. The array is configured such that thesequence and position of each member of the array is known. For example,a selection of genes that have potential to be expressed in certaindisease states may be arrayed on a solid support. Hybridization of alabeled probe with a particular array member indicates that the samplefrom which the probe was derived expresses that gene. Differential geneexpression analysis of disease tissue can provide valuable information.For example, if hybridization of a probe from a test (disease tissue)sample is greater than hybridization of a probe from a control (normaltissue) sample, the gene or genes expressed in the diseased tissue maybe a significant diagnostic indicator of a potential drug target.

Detection sensitivity is a limiting factor for effectively analyzingtest versus control samples such that gene expression, a geneticpolymorphism, or a gene mutation associated with the disease may berecognized. For the study of human genes using DNA microarrays,successful analysis of many disease states requires sensitive detectionto work with limiting sample quantities.

SUMMARY

The present invention relates to the discovery that detection of geneticdifferences, such as gene expression, genetic polymorphism, or genemutation, in diseased tissue relative to normal tissue, between tissuesat different developmental states, between individuals, and likecomparisons, is improved by the compositions and methods disclosedherein. The compositions and methods are useful for quantifying therelative amount of a component of a cell, where the component is anucleic acid (including a polynucleotide DNA or RNA), a polypeptide, aprotein, an antibody, and the like, by determining the amount of aparticular complex formed between the component (or its equivalent) anda target molecule on a support surface. For example, where the componentis a mixture of polynucleotides from a first biological sample and asecond biological sample, and the target molecule is a known or knowablenucleic acid sequence, the complexes are a hybridization complex betweenthe target molecule and the first and/or second polynucleotides. Thecomponent is preferably labeled as a detectable probe such that thecomplexes are distinguishable one from the other and the relativeamounts of the complexes may be determined as a measure of the amount ofthe component present in the first biological sample relative to thesecond biological sample.

In one aspect, the invention involves a microarray. The microarray ofthe invention comprises target molecules arrayed on a solid supportsubstrate in distinct spots that are at known, knowable or determinablelocations within the array on the support substrate. A spot refers to aregion of target molecule attached to the support substrate as a resultof contacting a solution comprising target molecule with the substrate.Preferably, each spot is sufficiently separated from each other spot onthe substrate such that they are distinguishable from each other duringdetection of complex formation. The microarray of the inventioncomprises at least one spot/cm², 20 spots/cm², 50 spots/cm², 100spot/cm², and greater densities, including at least 300 spots/cm², 1000spots/cm², 3000 spots/cm², 10,000 spots/cm², 30,000 spots/cm², 100,000spots/cm², 300,000 spots/cm² or more as the available technology allows.Preferably, the microarray of the invention comprises at least 2000spots/cm² to 25,000 spots/cm².

In an embodiment, the invention involves a microarray of biopolymers ona solid support substrate, wherein the substrate is silanized and thesilanization occurs with a silanizing agent in toluene as the solventand in the absence of acetone or an alcohol (such as methanol, ethanol,propanol, butanol, or the like). In a preferred embodiment thesilanizing agent is an organosilane and the solvent toluene issubstantially dry, wherein the drying is by standard techniques known inthe art. The organosilane may be any organosilane comprising an alkyl oraryl linker between the silicon atom and a reactive functionalitycapable of forming a covalent bond with a functionality on thebiopolymer or on another linker molecule useful in the invention.Preferably, the alkyl or aryl linker of the organosilane is from one to20 carbon atoms in length, preferably from 1 to 15, and most preferablyfrom 2 to 6 carbon atoms, inclusive. In a related embodiment, theorganosilane comprises a functionality that is capable of covalentlyattaching to the biopolymer directly or indirectly through anotherlinker molecule. The functionality on the organosilane may be, forexample, an epoxide, a halide, a thiol, or a primary amine (see, forexample, U.S. Pat. No. 6,048,695; U.S. Pat. No. 5,760,130; WO 01/06011;WO 00/70088, published Nov. 23, 2000). A useful organosilane forpracticing the invention is, for example, 3-aminopropyl triethoxysilane(APS) (see, for example, WO 01/06011; WO 00/40593; U.S. Pat. No.5,760,130; and Weiler et al., Nucleic Acids Research 25(14):2792-2799(1997)). According to this embodiment, the invention involves amicroarray comprising a biopolymer covalently attached to a substratewherein the substrate is silanized with a silanizing agent, and whereinthe substrate is reacted with the silanizing agent in toluene in theabsence of acetone or an alcohol, such as methanol, for example.

In another embodiment, the invention involves a microarray wherein thecovalent attachment of the biopolymer to the substrate is indirect, suchas, for example, through a linker molecule. Thus, according to thisembodiment, the invention involves a microarray comprising a biopolymerattached to a silanized substrate, wherein the microarray comprises alinker molecule between a substrate-attached silane and the biopolymer.According to a related embodiment, the microarray comprises abiopolymer, a silanizing agent, a multifunctional linker reagent, and asubstrate, wherein the biopolymer is attached to the multifunctionallinker reagent, the multifunctional linker reagent is attached to thebiopolymer and the silanizing agent, and the silanizing agent isattached to the substrate by a reaction in toluene in the absence ofacetone or alcohol. Preferably, the attachment between the biopolymerand the multifunctional linker reagent is covalent. Preferably, theattachment between the multifunctional linker reagent and the silanizingagent is covalent. Preferably, the substrate is glass and the reactionbetween the silanizing agent and substrate forms a covalent bond. In apreferred embodiment, the attachments, whether covalent or non-covalent,are sufficiently strong such that the biopolymer remains in its originalspot within the array during complex formation, washing steps, anddetection steps of microarray analysis. For an example of non-covalentattachment of nucleic acids and oligonucleotide probes in arrayhybridization reactions, see, for example WO 01/06011.

According to a related embodiment, the microarray of the invention isprepared by a method comprising silanizing a substrate, such as glass,with a silanizing agent in toluene in the absence of acetone or alcohol,followed by reacting a reactive functionality of the substrate-attachedsilanizing agent with a biopolymer to generate a biopolymer attached toa substrate. Preferably, the biopolymer is unmodified prior to reactingwith the substrate-attached silanizing agent. Alternatively, thebiopolymer is modified with a reactive functionality that reacts with afunctionality of the substrate-attached silanizing agent.

In a related embodiment, the microarray of the invention is prepared bya method comprising silanizing a substrate, such as glass, with asilanizing agent in toluene in the absence of acetone or an alcohol,followed by reacting the substrate-attached silanizing agent with amultifunctional linker reagent at one of its functionalities, followedby reacting another of the functionalities with a biopolymer.Preferably, the biopolymer is unmodified prior to reacting with themultifunctional linker reagent of the substrate-silanizingagent-multifunctional linker reagent linkage. Optionally, the biopolymeris modified with a reactive functionality that reacts with a reactivefunctionality on the multifunctional linker reagent of thesubstrate-silanizing agent-multifunctional linker reagent linkage. Thebiopolymer may be modified by any procedure appropriate for thebiopolymer of interest. For example, where the biopolymer is apolynucleotide, a reactive functionality may be introduced into thepolynucleotide during its synthesis or after it is synthesized.According to a non-limiting example disclosed herein, a primary amine isa reactive functionality introduced into the polynucleotide as aderivatized nucleic acid primer. Preferably, the multifunctional linkerreagent comprises two or more pendent chemically reactive groups(functionalities) adapted to form a covalent bond with a correspondingfunctional group on a substrate surface and adapted to form a covalentbond with a corresponding functional group on a target molecule.

According to a related embodiment, a substrate surface of a microarrayslide is derivatized with a silanizing agent and, optionally, with themultifunctional linker reagent to activate the microarray slide forimmobilizing the target molecule, wherein the activating comprises (1)silanizing the surface with an organosilane in toluene, preferably inthe absence of acetone or an alcohol (such as methanol, for example),wherein the organosilane comprises a functionality reactive with themultifunctional linker reagent, and wherein the activating furthercomprises immobilizing the multifunctional linker reagent on thesilanized surface by covalently reacting a first pendent reactive groupof the multifunctional linker reagent with the reactive functionality ofthe organosilane; (2) providing a solution comprising a target moleculehaving one or more functional groups reactive with a second pendentreactive group of the immobilized multifunctional linker reagent; and(3) attaching the target molecule to the substrate surface by contactingthe target molecule with the activated substrate surface and allowing afunctional group or the target molecule to form a covalent bond with thesecond pendent reactive group of the immobilized multifunctional linkerreagent.

In an embodiment of the invention, the target molecule of the microarrayis a nucleic acid, such as a polynucleotide of RNA, single stranded ordouble stranded DNA, a synthetic oligonucleotide, a peptide nucleic acid(PNA) in which the backbone is a polypeptide backbone rather than aribose or deoxyribose backbone, a polypeptide, a protein, an antibody, areceptor, a ligand, or like molecule that is detectable by its abilityto form a complex with another molecule, a detectable complexing agent.The polynucleotide may be from 5 nucleotides in length to and including10 kb in length. Preferably, the polynucleotide is from approximately100 bp to 5 kb, more preferably from 0.3 kb to 3 kb, and even morepreferably from approximately 0.5 kb to 2 kb. In an embodiment in whichthe target polynucleotide is PCR amplified double stranded DNA, thelength is preferably from 0.5 to approximately 2 kb. In an embodiment inwhich the target polynucleotide is a chemically synthesizedoligonucleotide, the length is preferably from approximately 50-1000nucleotides, 50-500 nucleotides, 50-200 nucleotides, 50-100 nucleotides.

In another embodiment, the invention involves a microarray of theinvention wherein the attached target molecule is a modifiedpolynucleotide and the modification is addition of an amine to thenative polymer. Preferably the amine is a primary amine and ispreferably at the 5′ end of the polynucleotide, but may be incorporatedelsewhere, depending on the constraints of polynucleotide preparation orthe needs of the microarray assay. Where a reactive group, such as aprimary amine, is preferred to be at the 5′ end of a polynucleotide, theprimary amine may be part of a primer that is enzymatically extended toproduce the primary amine-modified polynucleotide.

In still another embodiment, the substrate surface of the microarray ofthe invention comprises material selected from the group consisting ofpolymeric materials, glasses, ceramics, natural fibers, nylon andnitrocellulose membranes, gels, silicons, metals, and compositesthereof. Preferably the substrate is glass, more preferably a glassslide. Preferably the microarray substrate comprises at least one flatsurface comprising at least one of these materials. Optionally, thesubstrate is in a form of threads, sheets, films, gels, membranes,beads, plates, and like structures.

In another embodiment, the microarray of the invention is prepared bycontacting the target molecule with an activated substrate by atechnique from the group consisting of printing, capillary devicecontact printing, microfluidic channel printing, deposition on a mask,and electrochemical-based printing, wherein the contacting creates adiscrete target molecule-containing spot on the substrate (See, forexample, U.S. Pat. No. 5,700,637, U.S. Pat. No. 5,445,934, and U.S. Pat.No. 5,807,522 for particular methods of array formation, or Cheung, V.G. et al., Nature Genetics 21 (Suppl): 15-19 (1999) for a discussion ofarray fabrication). It is understood that various additional contactingtechniques are well known in the art or may be developed for depositinga target molecule to a solid support. Preferably, a technique is chosenthat is accurate, efficient, and economical for the user. In preferredembodiments where the target molecule is a modified or unmodifiedpolynucleotide, the target polynucleotide is contacted with thesubstrate in a solution, wherein the concentration of targetpolynucleotide in the solution is preferably the range of 0.1 μg/ul toand including 3 μg/μl. The pH of the solution is in the range fromapproximately pH 6-10, preferably approximately pH 6.5-9.7, morepreferably approximately pH 7-9.4. Preferably, the target polynucleotidesolution further comprises 500 mM sodium chloride, 100 mM sodium borate,pH9.3. Preferably, once the target biopolymer is contacted with thesubstrate under conditions according to the invention, the reaction israpid, preferably 1 hour or less, 30 minutes or less, 10 minutes orless, or five minutes or less. It was discovered as part of theinvention that allowing more time for the target polynucleotide to reactwith the activated slide improves detection sensitivity. For example,where the target polynucleotide is a double stranded or single strandedcDNA comprising a primary amine functionality and the activated slidesare prepared according to the present invention, the spotted slides areallowed to remain at ambient temperature and humidity for from 1-24hours, preferably about 5-18 hours, more preferably about 10-16 hours,and even more preferably about 12-14 hours before washing the slides toremove unreacted target molecule and other spotting solution componentsin preparation for hybridization and detection procedures.

According to the embodiment, the invention also involves blockingunreacted activating functionalities on the surface (e.g. unreactedsilanizing agent and/or unreacted multifunctional linker linkerreagent). Blocking reactions useful in the invention include washing theslides with water.

In another aspect, the invention involves an activated microarray slide,wherein the term “slide” refers to a solid support comprising at leastone substantially flat surface and the term “activated” refers to thepresence of reactive groups on the slide capable of reacting with amodified or unmodified target biopolymer according to the invention tocause the target biopolymer to be immobilized on the surface, such as bycovalent or non-covalent attachment. Preferably, the activated slidecomprises a silanized surface wherein the silanization occurred intoluene in the absence of acetone or an alcohol, such as methanol, forexample.

In a preferred embodiment, the activated slide further comprises amultifunctional linker reagent that is capable of linking thesurface-attached silanizing agent to the target biopolymer, therebybeing capable of immobilizing the target biopolymer on the microarrayslide. Preferably, the multifunctional linker reagent reacts first witha reactive functionality on the silanizing agent leaving at least onependent reactive group on the multifunctional linker reagent capable offorming an attachment with a functional group of the target molecule,wherein the attachment is non-covalent or covalent as long as the targetmolecule remains attached at its original location in the array. In apreferred embodiment, the surface comprises glass pretreated bysilanizing in toluene in the absence of acetone or an alcohol with anorganosilane comprising at least one reactive functionality that isreactive with at least one pendent reactive group of the multifunctionallinker reagent for immobilizing the multifunctional linker reagent.

In a preferred embodiment the target molecule is a polynucleotide andthe functional group of the target molecule is a hydroxy group, anepoxide, or an amine. Where the functional group on the targetpolynucleotide is an amine, it is preferably a primary amine.Optionally, the primary amine is preferably at the 5′ end of thepolynucleotide. In another preferred embodiment, the silane is anaminosilane, where the amino group is reactive with a multifunctionalreagent or a biopolymer.

In still another preferred embodiment, the silane is an organosilanecomprising a reactive group reactive with a multifunctional reagent orbiopolymer, wherein the organosilane is an alkyl silane and the alkylmoiety is selected from the group consisting or an ethyl-, a propyl-, abutyl-, a pentyl-, a hexyl-, a heptyl-, an octyl-, a nonyl-, and adecylalkyl moiety, and the reactive functionality of the organosilane iscovalently linked to the alkyl moiety. The alkyl moiety comprises acyclic portion. The organosilane may also comprise an aryl moietylinking the reactive functionalities to the silane. Where the reactivegroups on the silane and the target biopolymer are primary amines, thereactive groups on the multifunctional linker reagent are preferablythiocyanate groups reactive with primary amines.

Accordingly, an embodiment of the invention involves an activatedmicroarray slide comprising a silanized surface prepared by silanizingthe surface with an aminosilane in toluene in the absence of acetone oran alcohol, and a multifunctional linker reagent attached to the silane,wherein at least one pendent reactive group of the multifunctionallinker reagent is a thiocyanate moiety capable of reacting with anunmodified polynucleotide or a polynucleotide modified by theincorporation of a primary amine at its 5′ end.

In yet another aspect, the invention involves a method for preparing asolid support matrix to which nucleic acids are attached in making anucleic acid array. According to the invention, toluene is used as asolvent in silane-based modification by PDITC chemistry. The inventionderives from the discovery disclosed herein that DNA which is unmodifiedstill attaches to an activated glass solid support, such as a glassslide. The advantage of the present invention is that the use of tolueneas solvent in silanization of the glass, rather than acetone as thesolvent, reduces the fluorescent background and improves thesignal-to-noise ratio. In addition, the modified surface of the glassslide obtained by the method of the invention promotes the preparationof microarrays having improved nucleic acid spot morphology, such asreduced overlap with adjacent spots on a densely packed microarrayslide, and uniform distribution of the nucleic acid on the surfacecomprising the spotted region.

In another aspect, the invention involves a method of attaching a targetmolecule to a surface of a substrate, the method comprising providing anactivated microarray slide, wherein the activated slide comprises asilanized surface prepared by silanizing with an organosilane in toluenein the absence of acetone or an alcohol, and contacting a modified orunmodified biopolymer with the surface of the activated slide underconditions causing the biopolymer to covalently or non-covalently attachto the surface of the slide.

In an embodiment, the invention involves a reacting a multifunctionallinker reagent with a reactive group on the organosilane such that themultifunctional linker reagent is attached (covalently ornon-covalently) to the silane leaving at least one reactive group on themultifunctional linker reagent available to react with a modified orunmodified biopolymer. Preferably, the attachment of the multifunctionallinker reagent to the silane is covalent. Preferably, the reactivegroups on the multifunctional linker reagent are pendant in thatreaction between the linker and a modified or unmodified biopolymer isnot sterically hindered.

In an embodiment, the invention involves a method of attaching a targetmolecule to a surface of a substrate, wherein the method comprises firstproviding a solid support surface comprising at least one substantiallyflat surface. Next, the solid support surface is silanized with asilanizing agent in toluene in the absence of acetone or an alcohol,wherein the silanizing agent comprises a reactive functionality reactivewith a target biopolymer. The target biopolymer is then contacted withthe surface under conditions causing the target biopolymer to becomeattached to the silanizing agent on the surface, thereby immobilizingthe target biopolymer on the surface. Where the biopolymer isunmodified, the reactive group on the silanizing agent is reactive witha naturally occurring functionality on the biopolymer. Where the targetbiopolymer is modified, it is preferably modified with a reactive groupthat is capable of reacting with and forming an attachment to afunctionality on the silanized surface of the support.

In a related embodiment, the invention involves a method of attaching atarget biopolymer to a support surface of a substrate, wherein themethod is like that just described except that after silanizing thesurface, a multifunctional linker reagent is attached to the silanefollowed by attachment of the target biopolymer to the multifunctionallinker. Preferably, the multifunctional linker reagent comprises a firstreactive group that reacts with a functionality on the silane and asecond reactive group that reacts with a functionality on the targetbiopolymer. The reactive groups of the silane, the multifunctionallinker reagent and, optionally, a modified biopolymer are chosen toallow rapid and efficient reaction and attachment of the molecules tothe surface. Preferably, the silane is an aminosilane, the linker is adiisothiocyanate compound, and the biopolymer, if modified, is modifiedwith a 5′ primary amine. In a preferred embodiment, the silane is anorganosilane, such as 3-aminopropyltriethoxysilane. In another preferredembodiment, the multifunctional linker reagent is phenylenediisothiocyanate. Optionally, the target biopolymer is unmodified priorto reaction with the silane or the linker reagent.

In another aspect, the invention involves an improved method of nucleicacid (DNA and RNA) purification from tissue samples. The methodcomprises, in part, a modified cesium chloride purification useful fornucleic acid preparations from tissues or cell culture, for example. Thehighly purified RNA according to the invention, for example, is usefulfor the making of probes directly from RNA without a polyA+ purificationstep, which step causes substantial loss of starting RNA material. Themethod is also useful to re-purify commercially available RNAs toimproved detection sensitivity.

In one aspect, the invention involves improved methods for generatingfluorescently labeled sDNA probes from small quantities of nucleicacids, particularly ribonucleic acids. In mammalian tissue, for example,approximately 1% of the total RNA is messenger RNA/polyA+ RNA. BecausemRNA/polyA+ RNA is the material providing the initial template for DNAprobe synthesis, it is available in very small amounts against a complexbackground of non-messenger RNAs (ribosmal RNA, transfer RNA, and thelike). Consequently, the method of the invention for DNA probe synthesisprovides an advantage because the quantities of RNA useful as a templateaccording to the present method are 100-1000 fold less than the amountsuseful in previously known methods.

According to this aspect, the invention involves a method of preparing anucleic acid probe capable of forming a detectable complex with a targetmolecule, the method comprises isolating an amount of RNA from abiological sample; synthesizing a mixture of detectably labeled cDNAprobes complementary to the isolated RNA in the presence of a detectablylabeled deoxyribonucleotide; degrading ribonucleic acid with RNase;decreasing the average length of the labeled cDNA probes in thepreparation to be from approximately 0.5 kb to approximately 2 kb bylimited DNase digestion; and isolating the labeled cDNA probes.According to the invention, the isolated RNA is total cellular whichincludes messenger RNA. Preferably, the biological sample is selectedfrom the group consisting of a cell, a tissue sample, a body fluidsample, and a mixture of synthetic oligonucleotides.

In an embodiment, the invention involves a method for generatingfluorescently labeled sDNA probes using small quantities of totalcellular RNA, where the quantities are nanograms or picograms. Suchsmall amounts of total RNA are equivalent to low picogram or femtogramquantities of cellular messenger RNA, where mRNA is the actual templatefor reverse transcription to sDNA. Additional embodiments of theinvention include generating fluorescently labeled DNA probes from RNAisolated from cells, such as cells in tissue or in cell culture. Wherethe cells are from tissue, such as diseased human tissues, tumor cellsare microdissected nearby non-tumor cells in the diseased tissues.Tissue from which total RNA is isolated includes non-diseased anddiseased tissue and further includes fresh tissue, frozen tissue, andformalin-fixed paraffin-embedded tissue. According to the invention, theamount of isolated total cellular RNA is from approximately 0.01 pg toand including approximately 10 mg, 1 pg to and including 10 μg, 100 pgto and including 100 ng, and 500 pg to and including 10 ng.

In an embodiment of the method of preparing a cDNA probe, the inventioninvolves the additional steps of synthesizing double stranded DNA frommessenger RNA in the isolated total cellular RNA, followed bysynthesizing RNA complementary to the double stranded DNA. It isunderstood that cellular DNA may be isolated from the biological sampleand used as starting material for a DNA or cRNA probe according ot theinvention.

In another embodiment, the method of preparing a cDNA probe involveslabeling the synthesized cDNA probe by incorporating a detectablylabeled deoxyribonucleotide. Preferably, the labeled deoxyribonucleotideis dUTP. In a related embodiment the synthesizing of the labeled cDNAprobe is performed in the presence of labeled and unlabeled dUTP and inthe absence of dTTP.

Preferably, the detectable label is a fluorescent molecule and thedetection is by fluorescence emission. Other methods of detection may beused, including, but not limited to radioisotope labeling and detection,as well as mass spectrometry (see, for example, Marshall, A. andHodgson, J., Nature Biotechnology 16:27-31 (1998)).

Preferably, where the biological sample is a cell culture or tissuesample, the cells of interest from the culture or tissue arespecifically extracted from the biological sample generally independentfrom surrounding cells that of a different type or different diseasestate that are present nearby in the tissue or culture. Preferably, acontrol sample (e.g. a sample of normal tissue) comprises cells removedfrom the tissue source by laser capture microdissection, wherein thecell source is selected from the group consisting of untreated tissue,frozen tissue, paraffin-embedded tissue, stained tissue, and cellculture. Preferably, a test sample (e.g. a sample of diseased tissue)comprises cells removed from the tissue source by laser capturemicrodissection, wherein the cell source is selected from the groupconsisting or untreated tissue, frozen tissue, paraffin-embedded tissue,stained tissue, and cell culture.

In another aspect, the invention involves a method for generatingfluorescently labeled cRNA probes from small quantities of totalcellular RNA, where the quantity is nanograms or picograms. Such smallamounts of total RNA are equivalent to low picogram or femtogramquantities of cellular messenger RNA, where mRNA is the actual templatefor generation of double stranded DNA followed by transcription to cRNA.Additional embodiments of the invention include generating fluorescentlylabeled cRNA probes ultimately from RNA isolated from cells, such ascells in tissue or in cell culture. Where the cells are from tissue,such as diseased human tissues, tumor cells are microdissected nearbynon-tumor cells in the diseased tissues. Tissue from which total RNA isisolated includes non-diseased and diseased tissue and further includesfresh tissue, frozen tissue, and formalin-fixed paraffin-embeddedtissue.

In an embodiment, the invention involves a method of preparing a nucleicacid probe capable of forming a detectable complex with a targetmolecule, where the method comprises isolating an amount of RNA from abiological sample; synthesizing a mixture of detectably labeledcomplementary RNA probes by synthesizing double stranded DNA frommessenger RNA in the isolated RNA, followed by synthesizing RNAcomplementary to the double stranded DNA in the presence of a detectablylabeled ribonucleotide; and isolating the labeled cRNA probes.Optionally, sDNA is prepared by synthesizing cRNA complementary to thedouble stranded DNA, but in the absence of fluorescent deoxynucleotides,followed by synthesizing sDNA probes from the cRNA in the presence oflabeled fluorescently labeled deoxynucleotides and using random primers.Random priming controls the length of the sDNA probes. Preferably, theaverage length of the labeled sDNA probes is from approximately 0.5 kbto approximately 3 kb, preferably from approximately 0.5 kb toapproximately 2 kb. For cDNA probes, the average length is altered, ifnecessary, by mild Dnase digestion. For cRNA probes the average lengthof the labeled probes is decreased by mild RNase digestion or limitedfragmentation by resuspending the precipitated, labeled cRNA probes in40 mM tris-acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesiumacetate, followed by heating at 70° C. for 10 min. Preferably, theisolated RNA is total cellular RNA. Preferably, the biological sample isselected from the group consisting of a cell, a tissue sample, a bodyfluid sample, and a mixture of synthetic oligonucleotides.

In another embodiment, the method of preparing a cRNA probe involveslabeling the synthesized cRNA probe by incorporating a detectablylabeled ribonucleotide. Preferably, the ribonucleotide is UTP.Preferably the detectable label is a fluorescent molecule. In a relatedembodiment the synthesizing of the labeled cRNA probe is performed inthe presence of labeled and unlabeled UTP.

In another aspect, the invention involves a method for generatingfluorescently labeled sDNA (sense strand DNA) probes from smallquantities of total cellular RNA, where the quantity is nanograms orpicograms. Such small amounts of total RNA are equivalent to lowpicogram or femtogram quantities of cellular messenger RNA, where mRNAis the actual template for generation of double stranded DNA followed bytranscription to cRNA as an amplification step and without incorporationof label in the cRNA. To generate labeled sDNA probes, the cRNA isreverse transcribed in the presence of fluorescent nucleotides,preferably fluorescent dUTP nucleotides.

In still another aspect, the invention involves a method for generatingfluorescently labeled sDNA probes from total cellular RNA withoutamplification. According to the invention, total cellular RNA was usedas the starting material for first strand DNA synthesis. Labeled sDNAprobes are prepared by direct synthesis of a second strand DNA from thefirst strand using the Klenow fragment of DNA polymerase I.

According to the invention, the amount of isolated RNA useful for probesynthesis (cDNA, cRNA, or sDNA probes) is from approximately 0.01 pg toand including approximately 10 mg, 0.5 pg to and including 1 ng, 1 pg toand including 500 μg, 10 pg to and including 10 μg, 100 pg to andincluding 100 ng, and 500 pg to and including 10 μg.

According to the methods of preparing nucleic acid probes, the inventioninvolves deriving control nucleic acid probes from total cellular RNAfrom a control sample comprising a single or pooled mixture of samplesof similar tissue type, tissue origin, developmental stage, or the like.For example, the control sample comprises samples of normal tissue ofthe same organ from different donors or derived from the same tissuetype from the same or different donors. For example, in one embodiment,the invention involves pooling multiple epithelial tissues as a controlsample from which a control nucleic acid probe is derived for use indetecting gene expression or copy numbers in comparison with expressionor copy numbers in a test carcinoma. In a related embodiment, thecontrol sample is a mixture of cells from one or more cell cultures,where the cells are pooled prior to isolation of total cellular RNA. Acontrol nucleic acid probe generated from pooled cell cultures iscompared to a test nucleic acid probe in its ability to complex with atarget molecule. According to the invention, the test nucleic acid probemay also be derived from a mixture of test tissue cell samples or testcell culture samples.

In another aspect, the invention involves a method of preparing glassslides for application of nucleic acid in a microarray pattern, whereinthe method involves cleaning the slides with detergent and alkali;silanizing the slides with an organosilane in toluene in the absence ofacetone or an alcohol; optionally reacting the organosilane with amultifunctional linker reagent capable of reacting with a functionalgroup of the organosilane and a target molecule; followed by contactingthe activated surface (comprising the reactive organosilane attached tothe surface or, if present, the multifunctional linker reagent attachedto the organsilane) under conditions that cause the target molecule tobe attached to the surface by covalent or non-covalent attachment. Themethod also involves the steps of washing the silanized slides insolvents including toluene, methanol, water, and methanol to removeunreacted compounds and drying the slides after the attachment or theorganosilane, the multifunctional linker reagent, and the targetmolecule.

In an embodiment, the toluene is at least 50% of the solvent in thesilanizing step, preferably at least 80%, more preferably at least 90%,more preferably at least 95%, still more preferably at least 99%, andmost preferably the toluene is at least 99% of the solvent in thesilanization reaction mixture and is dried by standard techniques and ofstandard purity suitable for efficient silanization reactions andminimal background fluorescence during subsequent detection stepsaccording to the invention.

In another embodiment, the invention involves a method of attaching amodified target polynucleotide to a microarray solid support, whereinthe method comprises obtaining a nucleic acid primer comprising areactive group covalently attached to its 5′ end by a linker, whereinthe primer is complementary to sequences outside the targetpolynucleotide; amplifying the target polynucleotide by polymerase chainreaction to produce modified target polynucleotide comprising thereactive group; obtaining an activated microarray comprising on asurface a surface reactive group capable of reacting with the modifiedtarget polynucleotide reactive group, wherein the microarray solidsupport is pretreated by silanizing the surface with an organosilane intoluene; contacting the modified target polynucleotide with themicroarray solid support, whereby the modified target polynucleotidereactive group and surface reactive group react covalently attaching themodified target polynucleotide to the microarray solid support.Preferably, the modified target polynucleotide reactive group comprisesa primary amine and the surface reactive group comprises aisothiocyanate moiety.

In another aspect, the invention involves a method of analyzing abiopolymer target on a microarray, wherein the method comprisesproviding a microarray slide comprising a target biopolymer attached toa silanized substrate surface, prepared by silanizing with anorganosilane in toluene in the absence of acetone or an alcohol;contacting the attached target molecule with an agent capable of forminga detectable complex with the target molecule under conditions thatallow formation of a detectable complex; detecting formation of adetectable complex; determining the amount of a detectable complexformed.

In an embodiment, the agent capable of forming a detectable complexcomprises (1) a control mixture of nucleic acid probes comprising afirst detectable label, wherein the probes are prepared from nucleicacid isolated from a control sample, and (2) a test mixture of nucleicacid probes comprising a second detectable label, wherein the probes areprepared from nucleic acid isolated from a test sample, wherein thefirst and second detectable labels, and the nucleic acid molecules towhich they are attached, can be detectably distinguished one from theother for ease of determining the presence of, and optionally, therelative amounts of the probes in a mixture or the amounts of controland test probes forming complexes with a particular target molecule on amicroarray. The method further involves pooling the control probes andthe test probes; contacting the pooled probes with a target molecule ona microarray slide prepared according to the invention under conditionsthat allow the formation of specific detectable complexes between acontrol probe or a test probe; and comparing the amount of detectablecomplex formed between the target molecule and the control probesrelative to the amount of complex formed between the target molecule andthe test probes. Individual probes can also be singly hybridized to amicroarray to generate quantitative expression data that can be comparedto data from other singly hybridized or pooled probe hybridizedmicroarrays. Preferably the target molecule is a target polynucleotideand the probes are either cDNA probes cRNA probes, or cDNA probes, or acombination of these. Preferably the label is optically detectable, suchas by fluorescence emission. Preferably the complex formation betweenthe target molecule and the probes occurs in the absence of detergent,although a subsequent washing step optionally involves a solutioncomprising a detergent. In an embodiment of the invention, sodiumdodecyl sulfate (SDS) is eliminated from the hybridization solution inwhich a complex is formed between the target molecule and the probes. Inyet another embodiment, hybridization is performed in the presence of analkylammonium salt, DMSO and formamide to further improve complexformation.

In another aspect, the invention involves a method of hybridizing adetectable polynucleotide probe to a target polynucleotide on a supportsurface, the method comprising: (a) contacting the probe with denaturedtarget polynucleotide on the support surface in the absence ofdetergent; and (b) detecting formation of a complex between the targetpolynucleotide and the detectably labeled polynucleotide probe. In anembodiment of the invention, sodium dodecyl sulfate (SDS) is eliminatedfrom the hybridization step. In another embodiment of the invention,hybridization efficiency is improved by using a hybridization solutioncomprising formamide and one or more of an alkylammonium chloride(preferably tetrameythlammonium chloride, or tetraethylammoniumchloride, or both) and dimethylsulfoxide (DMSO).

According to the invention, the test sample and control sample differfrom each other according to one or more of developmental state, diseasestate, pre-disease state, cell type, sample source, and experimentaltreatment conditions. Optionally, according to the invention, thecontrol sample comprises a mixture of samples that differ from the testsample according to one or more of developmental state, disease state,cell type, sample source, and experimental treatment conditions.Optionally, according to the invention, the test sample comprises amixture of samples that differ from the control sample according to oneor more of developmental state, disease state, cell type, sample source,and experimental treatment conditions.

In an embodiment of the invention, the target molecule is apolynucleotide and the nucleic acid isolated from the test sample andthe control sample is RNA, and wherein the comparing provides a measureof target polynucleotide expression in the test sample relative totarget polynucleotide expression in the control sample. Preferably, therelative measure of target polynucleotide expression indicates a diseasestate in the test tissue sample, and the disease state is selected fromthe group consisting of all forms of cancer, cardiovasular disease,neurological disease, inflammation, and any disease that may becharacterized by an alteration in gene expression relative to anon-disease state. In a related embodiment, the relative measure oftarget polynucleotide expression indicates a pre-disease state in thetest tissue sample. In another related embodiment, the target moleculeis a polynucleotide and the nucleic acid isolated from the test sampleand the control sample is DNA, and wherein the comparing provides ameasure of number of copies of the target polynucleotide in cells of thetest sample relative to target polynucleotide copies in the controlsample, and the relative measure of the number of copies of targetpolynucleotide indicates a disease state or a pre-disease state in thetest tissue sample.

DESCRIPTION OF THE EMBODIMENTS

Definitions

As used herein, the terms “attached.” “attachment,” “bound,” and liketerms refer to a physical or chemical linkage between at least twomolecules. For example, where the attachment is between a targetmolecule and a substrate surface, the attachment is preferably acovalent chemical bond. Where the attachment is between a targetmolecule to be immobilized on a substrate surface and a reactive linkerreagent on the surface, the attachment is preferably covalent.Electrostatic, hydrophobic, hydrophilic, or other noncovalent chemicalbonds may form the attachment, however, if such noncovalent bondsprevent migration of the target molecule from its initial point ofcontact on the support surface. Where the binding is within a complexbetween a target molecule and an agent (a probe) capable of complexingwith the target molecule, the binding is preferably electrostatic,hydrophobic, hydrophilic, or other noncovalent binding.

As used herein, the term “biopolymer” refers to a target molecule ofinterest that may be attached to a substrate according to a procedureappropriate to the structure of the biopolymer. Optionally, thebioplymer is a nucleic acid sequence, including a single stranded ordouble stranded polynucleotide, where the polynucleotide may be RNA,DNA, or PNA (peptide nucleic acid, wherein the nucleotide backbone is apeptide backbone). Where the biopolymer is a protein, such as a ligand,a receptor, an antibody, cell surface protein, and the like, the probeis, for example, a receptor, ligand, antibody, polynucleotide, or otherbiopolymer or smaller molecule capable of forming a complex with thetarget protein. Preferably, the biopolymer is known, knowable,determinable, or otherwise identifiable.

As used herein, the term “detergent” refers to a surfactant useful forcausing or enhancing denaturation of target molecules as well asenhancing wetting of the support surface during hybridization.Non-limiting examples of detergents includes sodium dodecylsulfate(SDS), Triton X-100, Nonidet P-40, and Tween-20.

As used herein, the term “discernable,” or “distinguishable,” withregard to detection of a complex formed by a target molecule with acontrol probe versus a complex formed by a target molecule and a testprobe, refers to the ability to detect a control complex as differentfrom a test complex by direct visual detection or assisted detectionthrough the use of a detecting instrument. For example, a complexcomprising a control probe labeled with a first fluorescent dye isdiscernable from a complex comprising a test probe labeled with a secondfluorescent dye where the first and second dyes emit at differentwavelengths.

As used herein, the phrase “disease state” refers to an abnormal stateof a cell or a tissue, where the abnormal state in a living animal orplant results in illness or death. Non-limiting examples of a cell ortissue in a diseased state include all forms of cancer, cardiovasulardisease, neurological disease, inflammation, and any disease that may becharacterized by an alteration in gene expression relative to anon-disease state.

As used herein, the term “dye 488” refers to a dUTP- or UTP-derivatizedfluorochrome, where the fluorescent chromophore excites at a wavelengthof 488 nm and emits around a peak wavelength of 530 nm. The Alexa Fluor488 Dye (Molecular Probes, Inc.) is an example of such a dye. Commonlyused fluorescein dye also emits at this wavelength, the green region ofthe visible spectrum, and is useful in the invention. The preferred dyefor use in the present invention is the most intensely emittingchromophore available to the user, which is more photostable thanfluorescein, and which is relatively unaffected by variations in the pHrange used in microarray hybridization analysis (for example between pH4 to 10). In addition. Alexa Fluor Dye 488 is advantageous because ithas a narrower emission spectrum which results in reduced fluroescenceinteraction with dye 546, thereby allowing improved signal-to-noiseratios.

As used herein, the term “dye 546” refers to a dUTP- or UTP-derivatizedfluorochrome, where the fluorescent chromophore excites at a wavelengthof 546 nm and emits around a peak wavelength of 590 nm. The Alexa Fluor546 Dye (Molecular Probes, Inc.) is an example of such a dye. Commonlyused Cy3 dye and tetramethylrhodamine (TRITC and TAMRA) also emit atthis wavelength, the red region of the visible spectrum, and are usefulin the invention. The preferred dye for use in the present invention isthe most intensely emitting chromophore available to the user.

As used herein, a “glass slide,” with respect to microarray solidsupport, refers to a piece of planar silica-based glass of a size,shape, and thickness to allow convenient manipulation of the slideduring microarray preparation and subsequent microarray analyses.

As used herein, a “multifunctional linker reagent” refers to a moleculecapable of binding to another molecule, polymer, or surface while alsocapable of binding to still another molecule, polymer, or surface. Forexample, a linker molecule comprises at least two reactive groupscapable of such binding to two or more other molecules. According to theinvention, examples of linker molecules include an organosilane capableof binding to a surface (such as a glass surface) through an alkoxysilyl moiety, and capable of reacting with a target molecule or anotherlinker molecule. Another linker molecule may be a bifunctional reagentcapable of reacting with a reactive functionality on a surface-boundorganosilane as well as being capable of reacting with an unmodified ormodified target molecule.

As used herein, the term “normal tissue” refers to tissue in which nodiscernable disease is observed according to standard medical diagnosticmethods, or at least a disease state of a test sample is not present inthe control normal tissue sample.

As used herein, the term “nucleic acid” refers to a deoxyribonucleosideor ribonucleoside, or a deoxyribonucleotide or ribonucleotide polymer ineither single-stranded or double-stranded form. The term furtherencompasses nonnatural analogs of natural nucleotides, such as peptidenucleic acids.

As used herein, the term “oligonucleotide” refers to a single-strandednucleic acid sequence comprising from 2-1000 nucleotides in length,10-750 nucleotides, 20-500 nucleotides, 50-400 nucleotides, or 50-200nucleotides in length. An oligonucleotide may be chemically synthesizedby standard techniques in the art of nucleic acid synthesis. Suchtechniques included, but are not limited to solid phase synthesisfollowed by release of the oligonucleotide from the solid phase prior toattachment to a microarray slide, and solid phase synthesis on amicroarray slide (see, for example, U.S. Pat. No. 5,445,934).

As used herein, the phrase “pre-disease state” refers to an abnormalstate of a cell or a tissue, where the abnormal state in a living animalor plant may not be detectable. The pre-disease state in the animaldoes, however, predispose the animal to eventual development of adisease state. Non-limiting examples of a pre-disease state includeabnormal levels of genetic material, such as gene copy numbers, abnormalsequences of genetic material, such as disease-associated polymorphisms,changes in gene expression that frequently precede a disease state, aswell as genetic profiling of tumor subtypes (see, for example, Hacia, J.G., Nature Genetics 21(Suppl):42-47 (1999); Heiskanen, M. A. et al.,Cancer Research 60:41-46(2000), Pollack, J. et al., Nature Genetics23:41-46 (1999); DeRisij, et al., Nature Genetics 14:457-460 (1996);Berns, A., Nature 403:491-492 (2000); and Alizadeh, A. A. et al., Nature403:503-511 (2000); Marx, J., Science 289:1670-1672 (2000)).

As used herein, the term “probe” refers to an agent, preferably adetectably labeled agent, capable of forming a complex with a targetmolecule immobilized on a surface. Where the target molecule is apolynucleotide, the probe is another polynucleotide, a nucleic acidspecific binding protein or antibody, or other nucleic acid bindingmolecule. For example, the probe is another polynucleotide such as RNAor DNA or a peptide nucleic acid (PNA, nucleic acid having a peptidebackbone). Where the target molecule is a protein, such as a ligand, areceptor, an antibody, cell surface protein, and the like, the probe is,for example, a receptor, ligand, antibody, polynucleotide, or otherbiopolymer or smaller molecule capable of forming a complex with thetarget protein. Preferably, the complex formed between the targetmolecule and the agent is specific and detectably distinguishable fromcomplex formation with other target molecules in a microarray. It isnoted that the term “probe” is occasionally used to describe theimmobilized biopolymer attached to a microarray surface. For thepurposes of the present disclosure, the term “probe” will be used torefer to a labeled molecule capable of forming a complex with animmobilized molecule (the “target” as used herein) on a support surface.

As used herein, the phrase “reactive functionality at the 5′ end” of apolynucleotide, refers to a reactive functionality (chemically reactivemoiety of a chemical compound) attached directly or indirectly via alinker, where the site of attachment is within 50 bp, 20 hp, 10 bp, 5bp, or 2 bp of the 5′ end of the nucleic acid sequence. Preferably, thereactive functionality is within the 5′ terminal nucleotide, either onthe nucleotide base or on the deoxyribose.

As used herein, the term “silanizing,” with respect to activatingmicroarray slides, refers to reacting a silane with a substrate surfacesuch that the silane attaches to the substrate surface. According to theinvention, silanizing a microarray substrate surface refers to thereaction in which the silane reacts with a siloxy group on the surface.According to the invention, the silanizing occurs in toluene and in theabsence of acetone or an alcohol. The toluene of the silanizing reactionis preferably substantially dry (such as commercially available reagentgrade toluene). According to the invention, acetone or an alcohol maycontact the microarray slide during other, non-silanizing reactions orwashes, but contact with acetone is preferably limited to 3 hours orless, preferably 2 hours or less, followed by thorough drying to removethe acetone. Preferably, the surface comprises silica. More preferablythe surface is a silica-based glass. According to the invention, thesilane preferably comprises a plurality of reactive functionalities (orreactive groups), wherein at least one reactive group is capable ofreacting with the surface causing the silane to be attached to surface,and at least one other reactive functionality which is capable ofreacting with a reactive functionality of a target molecule, therebyattaching the target molecule to the silane and, ultimately, to thesubstrate surface. Optionally, the target molecule attaches to amultfunctional linker reagent that, in turn, attaches to the silane viareactive functionalities on the multifunctional linker reagent and thesilane. It is understood that the linker reagent may comprise multiplelinker reagent monomers.

As used herein, the term “spotting” or “tapping,” with respect todepositing a target molecule on a microarray substrate surface, refersto contacting the surface with a device, such as a microarray printingpin, containing a target molecule such that the target molecule isdeposited on the surface and is in contact with the surface of themicroarray. Preferably, the spotting or tapping is via a capillary orother tube (such as within the printing pin) capable of depositing asmall volume of solution comprising target molecule on the surface,wherein the volume is 1 μl or less, 100 nl or less, 10 nl or less, 5 nlor less, 2 nl or less, 1 nl or less, or 0.5 nl or less. Preferably thespot formed by depositing the target molecule solution on the surface isseparated from other spots on the microarray such that subsequenthybridization or other reaction on the array is not adversely affectedby reactions on neighboring or nearby spots. Preferably, the spot isfrom 50-500 microns, from 75-300 microns, or from 100-150 microns indiameter.

As used herein, the term “substrate” refers to a solid support to which,according to the invention, a target molecule is attached, eitherdirectly or indirectly, by coupling one or more linker molecules to thesubstrate and ultimately to the target molecule. Non-limiting examplesof substrate according to the invention include polymeric materials,glasses, ceramics, natural fibers, silicons, metals, and compositesthereof. The substrate has at least one surface that is substantiallyflat. As used herein, the phrase “substantially flat” with regard to asubstrate surface refers to a surface that is macroscopically planar formore convenient application of target molecules in a two-dimensionalarray. Alternatively, the substrate may have a spherical surface or anirregular surface to which a target molecule is attached and to whichtarget molecule a probe may be complexed for detection of suchcomplexes.

As used herein, the term “unmodified,” as used with respect to a targetbiopolymer such as target polynucleotide of the invention, refers to apolynucleotide that lacks reactive functionalities added or incorporatedinto a polynucleotide during or after its synthesis, isolation, or otherpreparation. Generally, according to the invention, a biopolymer'sreactive functionality, the addition of which modifies a biopolymer, isone that allows attachment of the biopolymer to a microarray substrate.A unmodified biopolymer, on the other hand, lacks such a functionalityadded for the purpose of attaching a target biopolymer to a surfacedirectly or indirectly through a linker molecule. Stated another way, anunmodified biopolymer is one in a native state wherein thefunctionalities (reactive or otherwise) that are present in the moleculeare native to a naturally occurring like biopolymer. Where an unmodifiedtarget biopolymer covalently attaches to a microarray slide, theunmodified biopolymer does so at functionalities typical of a naturallyoccurring biopolymer or a biopolymer as it is isolated from a cell.Where the unmodified biopolymer is an unmodified polynucleotide, such asRNA, DNA or PNA, the unmodified polynucleotide attaches to the substrateat functionalities typical of a naturally occurring nucleic acid base, apolynucleotide backbone, or a polypeptide backbone.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of microarray images generated using fluoroprobessynthesized by the method of the invention from 1-5 ng total RNA frommicrodissected colon tumor cells.

FIG. 2A is a photograph of microarray images generated usingfluoroprobes synthesized by the method of the invention from 5 μg totalRNA isolated from formalin-fixed paraffin-embedded liver tissue. FIG. 2Bis a photograph of microarray images generated using fluoroprobessynthesized by the method of the invention from 5 μg total RNA isolatedfrom fresh frozen adult liver. Probes generated from paraffin-embeddedstarting material were comparable in detection sensitivity to probesgenerated from fresh frozen tissue (compare FIG. 2A and FIG. 2B). FIG.2C is a photographic image of a microarray analysis from aformalin-fixed paraffin-embedded colon tumor, 4 μg total cellular RNAstarting material. FIG. 2D is a scatter plot of the fluorescenceintensities from microarray analysis of colon tumor RNA isolated fromthe same patient, a fresh-frozen sample (X axis) versus a formalin-fixedparaffin-embedded sample (Y axis).

FIG. 3A is a photograph of microarrays showing hybridization of probessynthesized from breast tumor RNA. FIG. 3B shows hybridization of probessynthesized from epithelial-tissue RNA pool reference sample. Ingeneral, gene expression is quantified by comparison of the intensityand wavelength emitted from each spot for test versus control samples.

FIGS. 4A, 4B, and 4C are photographs of microarrays showing successfuldetection of hybridized sDNA probes synthesized from various amounts oftotal cellular RNA starting material from an ovarian carcinoma cellline. The figures display the results of a 1-color analysis offluorescence intensity achieved on a microarray according to theinvention when the amount of total cellular RNA starting material waslimited to 200 pg (FIG. 4A), 20 pg (FIG. 4B), and 2 pg (FIG. 4C).

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. The examples are provided so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the compounds, compositions, and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g. amounts, temperature, etc.), but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. (° C.). Thedisclosures of all citations in the specification are expresslyincorporated herein by reference.

Example 1 Nucleic Acid Preparation for Microarray Analysis

The invention is useful for detecting the presence of nucleic acids inany mixture of nucleic acids. The present invention finds its preferreduse, however, in the detection and quantification of gene expression intissue samples, a medium in which detection of gene expression hasheretofore posed distinct challenges. The present invention solves thisproblem by providing a method of improving the detection limit for geneexpression in tissue samples.

Collecting Cells for Control or Test Samples: Microarray analysis allowsthe direct comparison of cellular states between test and controlsamples of cells, tissue, body fluids, and the like. Such comparisonsare optimized when the test or control sample comprises exclusively orsubstantially only the cells of interest. For example, a diseasedtissue, such as cancer tissue, frequently comprises cancerous cells thathave infiltrated an area of normal cells. Thus, a sample of canceroustissue will often contain a mixture of normal and diseased cells and mayalso include several cell types found in the tissue or associated withthe cancer, such as cells associated with the inflammatory and immuneresponses to cancer. Preferably, a sample comprises only those cellsimportant to the analysis. According to the present invention, it ispreferred that the test sample comprises a collection of cells collectedspecifically by cell type or other desired state such that contaminationof the sample by cells of a different type or state are excluded.

The technique of laser-capture microdissection (LCM) is preferred forcell collection (see, for example, Emmert-Buck, M. R. et al., Science274:998-1001 (1996); Simone, N. L. et al., Trends in Genetics 14:272-276(1998); Glasow, A. et al., Endocrine Research 24:857-862 (1998); WO002892 (priority date Nov. 5, 1998); Luo, L. et al., Nature Medicine5:117-122 (1999); and Arcturus Engineering, Inc., www.arctur.com, lastvisited Mar. 20, 2001). LCM was developed to provide a method forobtaining pure populations of cells from specific microscopic regions oftissue sections under direct visualization (Simone, N. L. et al. supra).For the purposes of the invention and the present examples, the cells ofinterest were transferred to a polymer film activated by laser pulses, atechnique that maintained the integrity of the RNA. DNA, and proteins ofthe collected cells. The transferred cells were used for the isolationof total cellular RNA for subsequent use in the preparation of controlnucleic acid probes and test nucleic acid probes. The LCM device usedfor the examples disclosed here was from Arcturus Engineering, Inc.,(Mountain View, Calif., USA).

Isolation and Purification of Nucleic Acids from Biological Samples: Thenucleic acid preparation method of the invention involves a cesiumchloride density gradient protocol. It is useful for collecting both RNAand DNA from tissue samples of limited size and from a variety of tissuesources including, but not limited to tumor tissue of epithelial origin.RNA obtained by this method is sufficiently pure to allow the directsynthesis of probes from the RNA and allows improved probe labeling.This method was found to particularly useful for isolating RNA fromtissues such as liver or fetal heart which are rich in contaminatingcarbohydrates. Additionally, the method of the invention is useful forpurifying commercially obtained RNAs, thereby allowing for improvedprobe synthesis and labeling of RNAs from commercial sources.

Purification of nucleic acids from tissue samples is provided as anexample of the method of the invention and its usefulness. Tissuesamples from normal tissue (or a pool of normal tissues) is designated“control tissue” or “control sample” herein. Tissue samples from adiseased tissue, such as tumor tissue, is designated “test tissue” or“test sample” herein. Unless otherwise indicated, the preparation ofcontrol and test samples is the same in the present example.

Tissue, either test or control tissue, was ground to powder in liquidnitrogen, followed by douncing 8-10 times in at least 10 volumes oflysis buffer (4M guanidine thiocyanate, 25 mM sodium citrate, 0.5%N-lauryl sarcosine) to provide a tissue lysate. For example, toapproximately 100 mg of tissue, approximately 1-2 ml of lysis buffer wasadded. The lysate was centrifuged at 12,000 rpm in an SS34 rotor(approximately 12,100×g; Beckman Instruments, USA) for 10 min. to removeinsoluble material. The clarified lysate was then layered on top of 5.7Mcesium chloride/50 mM EDTA pH 8 (designated “CsCl” for convenience) at avolume-to-volume ratio of 1:2.25 CsCl:lysate.

The CsCl:lysate preparation was centrifuged at 150.000×g for at least 12hours to sediment RNA from the suspension. For tubes compatible with aSW 55 rotor (Beckman Instruments, USA), 3.5 ml lysate was layered on 1.5ml CsCl for a total volume of 5 ml. When a TLS 55 rotor (BeckmanInstruments) was used for smaller samples of 50-200 mg tissue, 1.4 mllysate was layered onto 600 μl CsCl and centrifuged.

The lysate was removed and retained for DNA purification. The RNA pelletwas observed as a glassy precipitate at the bottom of the centrifugetube. After removing cesium chloride solution from the centrifuge tubeand washing the pellet with highly purified water, the RNA pellet wasresuspended in a volume of TE (10 mM Tris, 1 mM EDTA, pH 8-8.5)sufficient, to resuspend the pellet. Resuspension may be slow, requiring12 or more hours to resuspend large pellets in small volumes.

Resuspended RNA was extracted by standard phenol:chloroform extractiontechniques. The RNA was precipitated by the addition of 0.1 volume(relative to the aqueous layer) of 3M sodium acetate and 3 volumes ofethanol. The precipitate was washed with 70% ethanol, followed bywashing with 95% ethanol. The pellet was dried and resuspended in highlypurified water, such as double-distilled and deionized water or thelike.

Where the sample was cells in culture, the method of purifying nucleicacids was modified as follows. To cells harvested from a 10 cm cultureplate or a 15 cm plate, 2 ml or 3.5 ml, respectively, of the lysisbuffer was added. Lysate was collected using a syringe equipped with an18 gauge needle. Low-speed centrifugation at 12,000 rpm in an SS34 rotormay be omitted for the preparation of cultured cell lysate. Followingcollection of lysate, the procedure for nucleic acid purification fromcultured cells was the same as that for tissue samples.

The retained DNA-containing lysate was doubled in volume with highlypurified water. Material was extracted by standard phenol:chloroformextraction techniques leaving DNA in the aqueous later. DNA wasprecipitated by the addition of 0.7 volume isopropanol. The precipitatewas pelleted at 13,000 rpm in a SS34 rotor (Beckman Instruments), forexample, and mixed with a minimum amount of TE to resuspend the pellet.

The purified and resuspended RNA and DNA are useful for the preparationof probes for microarray analysis. The ability to isolate both RNA andDNA in a highly purified from a tissue sample is particularly useful inpermitting correlation and comparisons between the number of gene copies(as DNA) and the level of expression (as RNA), for example.

Example 2 Preparation of Microarray Probes

The protocol disclosed herein for the preparation of a microarray probeis useful to analyzing very small quantities of RNA as starting materialfor probe synthesis. The protocol is particularly useful to generatemixtures of cDNA probes or sDNA probes from tumor cells isolated fromheterogeneous tumor tissue by laser capture microdissection, forexample. The number of tumor cells thus isolated is usually quite small,yet as few as 100 cells, even 10 cells, and as few as one cell is asufficient source of RNA for gene expression analysis due to thesurprising sensitivity available using the compositions and methods ofthe invention. The present method is also useful for probe synthesisusing RNA isolated from non-microdissected cells, but is generally,although not exclusively, most useful when the quantities of RNA arelimiting. The probes generated by the method of the invention arereliably sensitive even when the amount of RNA starting material is verysmall. For example, the invention relates to probe synthesis from aslittle as 2 pg-10 ng isolated total cellular RNA, which representsapproximately 20 fg-100 pg messenger RNA, an amount that isapproximately 1000-fold less than currently available techniques cananalyze.

According to the present invention, two variations for probe synthesisare disclosed, where the variations depend on the amount of isolatedtotal cellular RNA available. For quantities of total RNA from 500 ng-5μg, inclusive, a direct labeling protocol is used. For quantities of RNAas small as 500 pg-10 ng of total RNA, probes are generated by a singleround of amplification by in vitro transcription. For extremely smallamounts of total cellular RNA (e.g. 0.01-10 pg total cellular RNA,preferably about 1-10 pg, more preferably about 1-2 pg, equivalent tothe total RNA from a single cell), the initial amplification by in vitrotranscription may be performed as described, or performed for a longerincubation period (e.g. for 12 hours), or performed twice to generatesufficient material for sDNA probe or cRNA probe synthesis. For eachembodiment of the invention, cDNA probe, sDNA probe, or cRNA probesynthesis involves the incorporation or fluorochromes.

Before cDNA probe, sDNA probe, or cRNA probe synthesis, the RNA may bepurified by micro-CsCl centrifugation or by direct precipitation ofunquantified nucleic acid. For example, these purification protocolswere particularly useful when working with microdissected tissuesamples.

This example discloses the use of commercially available modifiedfluorescent dyes (the Alexa series of fluorescent dyes, MolecularProbes, Inc., Eugene, Oreg., USA) in a 2-color or one-color microarrayanalysis based on cDNA probes prepared directly by reverse transcriptionof isolated RNA purified by the method disclosed in Example 1.Similarly, cRNA probes and sDNA probes were prepared with anintermediate step of double stranded DNA synthesis from isolated RNA,followed by transcription, then, where a sDNA probe is desired, bysynthesis of a labeled DNA probe using reverse transcriptase, labeleddeoxyribonucleotides, and random primers. The method of probepreparation disclosed in this example is robust and highly sensitive,allowing the user to begin with as little as 500 pg-10 ng total RNA.

Preparation of Labeled DNA Probes:

The following procedures disclose non-limiting examples of methods ofpreparing a detectably labeled DNA probe for use in the presentinvention. In each example of probe synthesis, the starting material wastotal cellular RNA isolated from a tissue sample. As these examplesdemonstrate cDNA probes were prepared from RNA with no intermediateamplification or only 1 or 2 rounds of amplification sDNA probes wereprepared by reverse transcription from unlabeled cRNA, sDNA probes werealso prepared from larger amounts of starting total cellular RNA bydirect second strand synthesis with label incorporation, cRNA probeswere prepared from cDNA.

Problems to be Solved in Developing an Improved Method of PreparingLabeled Nucleic Acid Probes:

Detection sensitivity relies, in part, on the ability to generate amaximally labeled (“hot”) probe without exceeding the solubility limitsfor the DNA/chromophore complex. The solubility of the DNA/chromophorecomplex is affected by probe labeling density and probe length. Labelingdensity is defined as the number of chromophores per specified DNAfragment length. Labeling density was found as part of the invention tobe correlated with total labeling efficiency, and therefore, correlatedwith the ratio of labeled probe to unlabeled probe. This ratio isreadily estimated by probe intensity visualized on a nucleic acidsequencing gel. This technique was useful for evaluating the probes forapproximate labeling density, molecular weight or fragment length. In arelated observation, it was found that probe solubility was inverselycorrelated with labeling efficiency, i.e. as the number of fluorochromeswas incorporated into a probe, its solubility decreased. Thus, thevisualization of labeling efficiency on a sequencing gel also providedan indirect estimation and prediction of probe solubility.

The length and, hence the molecular weight, of the probes was controlledby mild DNase digestion. Preferably the DNase digestion is performed fora time and under conditions that yield an average probe length of lessthan 5 kb, more preferably in the range from 0.5 kb-2 kb, inclusive,after digestion. Gel electrophoresis may be used to evaluate the degreeof probe digestion. Redigestion by DNase can be performed if the averageprobe length is longer than the target average length.

Probes were evaluated for labeling density on an ABI 373A DNA sequencer(Applied Biosystems, Inc., USA) or other phosphoimaging or fluorescentimaging device. The use of fluoroscein- and rhodamine-related dyes wasuseful because the different emission wavelength of each dye allowedseparate detection of the labeling density for each dye. Labelingdensity was estimated by correlation with ratio of labeled to unlabeledprobe, such that the fluorescence intensity of the probe mixture on asequencing gel provides an indication of the labeling density. Otherdyes are, of course, useful in the method of the invention. Preferably,the dyes have emission maxima that do not directly overlap and allow theseparate and quantitative detection of chromophores in aprobe/microarray complex.

The solubility of a labeled probe was determined directly using a chargecoupled imaging device (a “CCD imager”). Solubility was also predictedby correlation with labeling density (e.g. the ratio of labeled tounlabeled probe) because an increased amount of label incorporationincreases the fluorescent intensity of the probes, but also increasesinsolubility. A suitable probe intensity as assessed by acrylamide gelelectrophoresis on an ABI373A DNA Sequencer (photon multiplier tubevoltage setting of 750-780 volts) includes visible, but non-saturating,fluorescent signal (100-4000 fluorescence units by the GeneScan softwarepackage, Applied Biosystems) on loading 0.5% of 488-labeled probes and5% of 546-labeled probes.

Detection sensitivity also relies on adjusting the stoichiometry ofchromophore and template nucleic acid to maximize probe labeling. It wasfound as part of the present invention that, during cDNA synthesis byreverse transcription from template RNA, that the unlabeled dNTPs of thereaction mixture should include unlabeled dUTP, instead of dTTPtypically required in standard procedures. The substitution of dUTP fordTTP improves efficiency of the mRNA labeling reaction because unlabeleddUTP competes less effectively than unlabeled dTTP for incorporation byreverse transcriptase, thereby increasing the number of chromophoresincorporated into a probe.

As another method of improving detection sensitivity, the presentinvention contemplates use of ribonuclease (RNase), rather than commonlyused alkali, to degrade the parent mRNA strands. It was discovered aspart of the present invention that the omission of alkali in mRNAdegradation was helpful because alkali substantially decreases thefluorescence emission of dye 488, one of the chromophores useful in theinvention.

Preparation of Labeled DNA Probes:

While the present example discloses a method for preparing a DNA probefrom RNA, it is also contemplated that DNA probes from RNA or DNA may beprepared based on the disclosure provided herein for related oralternative applications.

According to the invention, RNA strand extension was an initial step incDNA probe synthesis. A basic technique for RNA strand extension isavailable from differential display reverse transcriptase PCR(DDRT-PCR). In that technique, total cellular RNA is primed for firststrand reverse transcription with an anchoring primer composed ofoligo-dT and any two of the four deoxynucleosides (DDRT-PCR; see, Liangand Pardee, Science, 257:967-971 (1992) and Russell, D. W. and Thigpen,A. E., U.S. Pat. No. 5,861,248, issued Jan. 19, 1999). In one embodimentof the present invention, RNA strand extension uses an oligo-dTVN primerfor extension by a reverse transcriptase, such as Moloney MurineLeukemia Virus reverse transcriptase (MMLV-RT) in the presence or dATP,dGTP, dCTP, dUTP, and chromophore-labeled dUTP, and other components asdisclosed, infra. The present invention differs from DDRT-PCR, however,in that no amplification or only one round of amplification of the RNAor cDNA is performed. The methods disclosed herein improve detectionsensitivity to such a surprising extent that detection and quantitationof gene expression may be performed on very small mRNA samples withoutthe need for PCR-based or additional T7-based amplification or with onlyone round of linear amplification. As a result, the methods are rapid,convenient, and sensitive relative to existing methods.

Preparation of sDNA Probe

Detectably labeled sDNA probes were generated from 1 pg-10 ng total RNA.Because of the small amount of starting material, the present embodimentinvolves a single round of amplification prior to incorporation ofchromophore as disclosed in the following procedure. The term “sDNA”refers to DNA generated from total cellular RNA by first and secondstrand cDNA synthesis, followed by one round (or optionally two rounds)of cRNA synthesis to amplify the nucleic acids sequences, followed bysDNA synthesis by reverse transcription of the cRNA in the presence of adetectably labeled dNTP.

First Strand Synthesis: Into each sample reaction vial was added: 10 ngpurified total cellular RNA (isolated according to Example 1); 2 μgoligo-dTVN-T7 primer (oligo-dT refers to an oligomer of 18 dT residuescomplementary to poly-A tails of mRNA; V refers to nucleotides dA, dC,and dG; N refers to dA, dC, dG, and dT, and “T7” indicates that theoligo comprises the T7 promoter sequence,5′-GAATTCTAATCGACTCACTATAGT₁₈-3′ (SEQ ID NO:1), at the 5′ end of theoligo); and 0.8 μl dNTP mix (500 μM each of dATP, dGTP, dCTP, and dTTP).The samples were heated to 65° C. for 3 min., cooled on ice, and left atroom temperature for 10 min to anneal the primer to mRNA in the totalcellular RNA mixture. To each sample was added 4 μl 5× reaction buffer(250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂); 0.5 μl RNase Block;1 μl Superscript II; and 200 U Superscript reverse transcriptase (LifeTechnologies, Madison, Wis., USA) in a final volume of 20 μl. Thesamples were allowed to incubate at 42° C. for 1 hour to extend thefirst cDNA strand.

Second Strand Synthesis: To each sample vial from the First StrandSynthesis reaction, the following reagents were added: 91 μl DEPC water;30 μl 5× reaction buffer, supra; 3 μl 10 mM dNTPs; 1 μl E. coli DNAligase; 4 μl E. coli DNA polymerase; and 1 μl E. coli RNaseH. Thesamples were incubated at 16° C. for 2 hours.

In a related procedure, the reaction volume was reduced and the Klenowfragment of DNA polymerase I is used for an improved yield of doublestranded DNA and subsequently sDNA probe. To each sample vial from theFirst Strand Synthesis reaction, the following reagents were added: 18.1μl DEPC water; 10 μl 5× second strand buffer (Life Technologies); 1 μl10 mM dNTPs; 0.3 μl E. coli DNA ligase (10 U/μl); 0.3 μl E. coli DNApolymerase I Klenow fragment (50 U/μl); and 0.3 μl E. coli RNaseH (2U/μl), for a total volume of 50 μl. The samples were incubated at 12° C.for 2 hours.

The resultant double stranded cDNA was partially purified byphenol:chloroform extraction. The cDNA was then precipitated by theaddition of 85 μl 7.5 M ammonium acetate and 650 μl cold ethanol(approximately 0° C.) and 1 μl linear polyacrylamide, a nucleic acidcarrier for precipitation (Ambion, Inc.). For the smaller volumereaction disclose above, the volumes were adjusted such that 29 μl 7.5 Mammonium acetate and 220 μl cold ethanol (approximately 0° C.) and 1 μllinear polyacrylamide were added. A cDNA pellet was collected, washedand dried by standard techniques.

Amplification by Transcription from cDNA: A single round of linearamplification is preferred when only small amounts of total cellular areavailable. Amplification is achieved by transcribing mRNA from thedouble stranded cDNA generated by first and second strand synthesis,supra. When only very small quantities of total cellular RNA wereavailable from biological samples, (e.g. 1-20 pg of total RNA), thereaction was optionally followed as described, or the transcriptionreaction was allowed to continue overnight, or two rounds of linearamplification were performed. The following procedure describes a singleround of linear amplification.

The double stranded cDNA was resuspended in 20 μl 1× T7 TranscriptionReaction Buffer (Ambion, Austin, Tex., USA; T7 Megascript™ Kit, catalogno. 1337). To the resuspended cDNA were added the following components:8 μl DEPC water; 2 μl each of 75 μM solutions of ATP, GTP, CTP, UTP; 2μl 10× Buffer (Megascript™ Kit, Ambion, Inc.); 2 μl 10× T7 RNApolymerase. The samples were incubated at 37° C. for 5 hours. Overnightincubation under these conditions increased the yield. The reactionswere stopped by the addition of 15 μl sodium acetate stop buffer (7.5 Msodium acetate), 115 μl DEPC water and extraction withphenol:chloroform. The nucleic acids were precipitated with an equalvolume of isopropanol.

Incorporation of Fluorochromophore: Label may be incorporated into cDNAsynthesized directly from mRNA present in total cellular RNA if 500 ng-5μg or more is available. For direct cDNA synthesis from total cellularRNA, the following fluorochromophore incorporation procedure is useful.When less than 500 ng total cellular RNA was available, linearamplification as disclosed, supra, is preferred.

For probe synthesis after amplification, 5 ng-100 ng of cRNA pellet wassuspended in 1×. First Strand Reaction Buffer, supra. To the resuspendednucleic acids were added the following components: 1 μg random hexamers;0.8 μl nucleotide mix (10 mM each dATP, dGTP, dCTP, and 7 mM dUTP); andDEPC water to bring the volume to 13.5 μl. The nucleic acids weredenatured and the hexamers annealed by placing the samples at 65° C. for3 min., chilling on ice, and then annealing at room temperature for 10min. Optionally, from 100 ng to 10 μg random hexamers are added to thereaction.

Next, fluorochromophore was incorporated as follows: To each vial wereadded the following: 4 μl RNase Block; either dUTP-fluorophore (6-12 μMAlexa 546-dUTP or 2540 μM Alexa 488-dUTP); and 1 μl MMLV reversetranscriptase (200 U). The reaction was incubated for 1 hour at 42° C.in the dark. The sDNA probes generated from control and test sampleswere labeled with different, detectably distinguishable chromophores.For example, the control probes were labeled with dye 546 and testprobes were labeled with dye 488.

The parental RNA strands were removed from the sDNA probe mixture byRNase digestion according to the following protocol. Each reaction vialwas heated to 95° C. for 1 min., followed by chilling on ice to denaturethe DNA and RNA strands. To each reaction vial, was added 1 μl dilutedRNase (500 μg/ml diluted 1:50 in water; Boehringer-Mannheim). The RNasedigestion was allowed to continue for 15 min. at 37° C. The reactionvials were then placed on ice until the next step could be performed.

As an aspect of the invention, the average sDNA probe length wascontrolled by the stoichiometry of random hexamer primer to cRNA suchthat the average probe length was 0.5-2 kb. As the ratio or randomprimers to cRNA increased, the average probe length (related to averageprobe molecular weight) decreased.

Preparation of Labeled cDNA Probe Directly from Total Cellular RNA Inanother aspect, the invention involves a method of preparing labeledcDNA probes directly from total cellular RNA by incorporating detectablylabeled dNTPs in the reaction mixture for first strand synthesisaccording to the first strand synthesis procedure disclosed, supra.

According to this method, first strand cDNA synthesis with direct labelincorporation was performed as follows. Into each sample reaction vialwas added: 1-10 μg purified total cellular RNA (isolated according toExample 1); 2 μg oligo-dTVN-T7 primer (oligo-dT refers to an oligomer of18 dT residues complementary to poly-A tails of mRNA; V refers tonucleotides dA, dC, and dG; N refers to dA, dC, dG, and dT, and “T7”indicates that the oligo comprises the T7 promoter sequence.5′-GAATTCTAATCGACTCACTATAGT₁₈-3′ (SEQ ID NO:1), at the 5′ end of theoligo); and 0.8 μl dNTP mix (500 μM each of dATP, dGTP, dCTP, and dTTP).The samples were heated to 65 C for 3 min., cooled on ice, and left atroom temperature for 10 min to anneal the primer to mRNA in the totalcellular RNA mixture. To each sample was added 4 μl 5× reaction buffer(250 mM Tris-HCl. pH 8.3, 375 mM KCl. 15 mM MgCl₂); 0.5 μl RNase Block(Stratagene); 1 μl Superscript II; and 200 U Superscript reversetranscriptase (Life Technologies. Madison, Wis., USA) in a final volumeof 20 μl. The samples were allowed to incubate at 42° C. for 1 hour toextend the first cDNA strand.

The average cDNA probe length was next adjusted with limited Dnasedigestion. The cDNA reaction volume in each vial was adjusted to 50 μlwith 10 mM MgCl₂ and chilled on ice. A dilute DNase I solution wasprepared comprising 1 part DNase I (10,000 U/ml; Boehringer-Mannheim) in5000 parts 20 mM Tris buffer, pH 8.0. The final dilution of DNase I wasapproximately 2 U/ml. A 2 μl aliquot of diluted DNase 1 (2 U/ml) wasadded to each vial containing cDNA probe labeled with dye 546, and a 4μl aliquot of diluted DNase I was added to vials containing cDNA probelabeled with dye 488. The DNase conditions may be varied as necessary toadjust for different chromophores and input cDNA. The vials wereincubated at 12° C. for 30 min. Next 5 μl 250 mM EDTA pH 8.0 was addedto each vial. DNase I was inactivated by heating each vial to 65° C. for15 min. The labeled cDNA probe was separated from the proteins bystandard phenol:chloroform extraction followed by purification of theaqueous layer over a G50 spin column (Pharmacia). To each aqueous eluatefrom the spin columns was added a 3 μl aliquot of a 10×SSC solution. ThecDNA probe pellet was dried and resuspended in a 6 μl aliquot of 50:50formamide:water solution for at least 3-4 hours at room temperature inthe dark. Once a flurochromophore is incorporated into a probe, theprobe is preferably kept in the dark at 0° C. or below until ready touse. The resuspended labeled cDNA probe is useful for hybridization tomicroarrays as disclosed herein.

Preparation of Labeled sDNA Probe Directly from First Strand cDNA Inanother aspect, the invention involves a method of preparing labeledsDNA probes directly from cDNA without intermediate cRNA synthesis(without amplification). The probes are prepared by second strand sDNAsynthesis with simultaneous incorporation of label. Average probe lengthis controlled by the use of random primers in the final labeling step.The method is similar to the method for preparation of labeled cDNAprobes with the following modifications. The probe labeling involvesdouble stranded cDNA preparation as disclosed, supra, followed bylabeling of sense strand DNA (sDNA) using fluorescentdeoxyribonucleotides and random primers. The unlabeled first strand DNAis synthesized using a biotin-labeled primer and can be removed, toavoid competition in hybridization, using streptavidin. A non-limitingexample of the method follows.

RNA isolation from samples: RNA was prepared from frozen tissue, samplesisolated by laser capture microdissection (LCM), or from tissue storedin RNAlater reagent (Ambion, Austin, Tex., or Qiagen, Valencia, Calif.).Samples were homogenized with a rotor-stator tissue homogenizer (IKALabortechnik, Staufen, Germany, or Brinkman Instruments, Westbury, N.Y.)in RLT buffer according to the RNeasy Mini or Midi RNA purification kits(Qiagen, Valencia, Calif.). Purified RNA was quantified by measuringoptical absorption at 260 nm in a UV spectrophotometer (Shimadzu,Pleasanton, Calif.). For RNA purified from small amounts of tissue (<1μg of tissue, or LCM tissue sample) the RiboGreen RNA quantitation assay(Molecular Probes, Eugene, Oreg.) was used with a fluorescencemicroplate reader (Molecular Devices, Sunnyvale, Calif.).

First Strand cDNA Synthesis: First strand cDNA was synthesized from0.5-5 μg of total RNA using Superscript reverse transcriptase asdescribed by the manufacturer (Life Technologies, Rockville, Md.) using5′-biotin labeled (dT)₁₈VN, where V=G, A, or C and N=G, A, T, or C. RNAwas then digested in 10 ng of DNase-free RNase A (Roche MolecularBiochemicals, Indianapolis, Ind.) 37° C. for 15 minutes. The reactionwas extracted with water saturated phenol:chloroform:isoamylalcohol(49:49:2). Linear acrylamide (Ambion, Austin, Tex.) was added to a finalconcentration of 18 ng/ml. One-tenth volume of 3 M sodium acetate pH 4.8was added and cDNA was precipitated by the addition of an equal volumeof ice cold isopropanol. Samples were incubated at −20° C. for 20minutes, centrifuged at 14,000 rpm for 20 minutes at 4° C., and thesupernatant was aspirated from the clear pellet which was vacuum dried.

Second Strand cDNA Synthesis of Incorporation of Fluorochrome: Secondstrand cDNA was synthesized in 20 μl reaction using 2 Units of theKlenow fragment of DNA polymerase I (Life Technologies, Rockville, Md.),1 to 50 μg or p(dN)₆ (Life Technologies, Rockville, Md.) or other randomsequence oligonucleotide of 7 to 9 bases, 100 μM each of dGTP, dCTP, anddATP, and a combination of dTTP and Alexa488-dUTP (Molecular Probes,Eugene, Oreg.) to a final concentration of 100 mM. The dTTP toAlexa488-dUTP ratio may vary from 100:1 dTTP to Alexa488-dUTP to 100%Alexa488-dUTP. Alexa 546-dUTP and Alexa 594-dUTP may also be used withthis protocol. NaCl may be added in addition to the standard workingconcentration of 50 mM, increasing in concentration up to approximately150 mM. The reaction included reaction buffer components supplied by theenzyme supplier (Life Technologies. Rockville, Md.). Reactions wereinitiated by first heating the reaction mixture to 95° C. for 5 minutes,then quickly chilling it on ice, followed by the addition of Klenowenzyme. The reaction was incubated at temperatures ranging from 12° C.to 37° C. for 1 to 18 hours. Reactions were stopped by addition of EDTAto 25 mM, heated at 95° C. for 5 minutes and quickly chilled on ice. Thebiotin-tagged (−) strand cDNA is separated from random-primed (+)-strandlabeled cDNA using streptavidin-paramagnetic particles (SA-PMP)(Promega, Madison, Wis.). SA-PMPs were prepared by washing 3 times in0.5×SSC and once in 10 mM Tris, 1 mM EDTA, pH 7.5. The labeled cDNAreaction was incubated with the SA-PMPs for 10 minutes at roomtemperature, and the supernatant was removed from the SA-PMPs on amagnetic stand. The resulting labelled (+) strand cDNA was extractedwith water saturated phenol:chloroform:isoamylalcohol (49:49:2),purified over a G-50 spin column (Pharmacia), and vacuum dried beforeusing in a hybridization reaction.

Preparation of Labeled cRNA probes: Preparation of labeled cRNA probesis performed, according to the invention, by direct incorporation offluorochromophore-labeled ribonucleotides into cRNA followed byadjustment of average probe length. The method is similar to the methodfor preparation of labeled cDNA probes with the following modifications.The cRNA probe synthesis begins from the step of double stranded cDNApreparation as disclosed, supra.

Double stranded cDNA prepared was resuspended in 20 μl 1× T7Transcription Reaction Buffer (Ambion, Austin, Tex., USA; 17 Megascript™Kit, catalog no. 1337). To the resuspended cDNA were added the followingcomponents: 8 μl DEPC water; 2 μl each of 3.75 mM solutions of ATP, GTP,CTP, UTP; 2 μl 10× Buffer (Megascript™ Kit, Ambion, Inc.); 2 μl 10× T7RNA polymerase. To each vial were added the following: 4 μl RNase Block;either UTP-fluorophore (60 μM Alexa 546-UTP (preferably in the range of30-120 μM, inclusive) or 300 μM Alexa 488-UTP (preferably in the rangeof 200-400 μM, inclusive)). The samples were incubated at 37° C. for 5hours, or overnight for further improvements in yield. The reactionswere stopped by the addition of 15 μl sodium acetate stop buffer (7.5 Msodium acetate), 115 μl DEPC water and extraction withphenol:chloroform. The nucleic acids were precipitated with an equalvolume of isopropanol. Using this procedure, the cRNA probes weregenerated from control and test samples and were labeled with different,detectably distinguishable chromophores. For example, the control probeswere labeled with dye 546 and test probes were labeled with dye 488.

The preferred average cRNA probe length was from 0.5 kb to and including3 kb. The average probe length and labeling density of the cRNA probeswas estimated by observing the probes on a sequencing gel such as an ABI373A gel (Applied Biosystems, USA). The labeling density was estimatedaccording to an observed correlation between an increase in labelingdensity and the ratio of labeled to unlabeled cRNA probe.

If it was determined that the average length should be reduced, thelabeled cRNA probe length was adjusted by resuspending the precipitated,labeled cRNA probes in 40 mM tris-acetate, pH 8.1, 100 mM potassiumacetate, 30 mM magnesium acetate. The resuspended, labeled cRNA probeswere incubated at 70° C. for 10 min. Optionally, mild RNase digestionmay be used to decrease the average length of the cRNA probes. It isunderstood that reaction conditions may vary and are readily adjusteddepending on the beginning average probe length and label density. Oncea flurochromophore is incorporated into a probe, the probe is preferablykept in the dark at 0° C. or below until ready to use. The resuspendedlabeled cDNA probe is useful for hybridization to microarrays accordingto the invention.

The preferred average DNA probe, sDNA probe, or cRNA probe length wasfrom 0.5 kb to and including 3 kb, preferably from 0.5 kb to andincluding about 2 kb. The average probe length and labeling density ofthe sDNA probes was estimated by observing the probes on a sequencinggel such as an ABI 373A gel (Applied Biosystems, USA). The labelingdensity was estimated according to an observed correlation between anincrease in labeling density and the ratio of labeled to unlabeledprobe. The stoichiometry of random hexamers to cRNA is the preferredmethod for controlling the average sDNA probe length. The average lengthof cDNA probes is preferably adjusted by mild Dnase digestion asdisclosed herein.

Design of Controls for Microarray Analysis: In the present examples,carcinomas, cancers of epithelial tissue, were studied for geneexpression relative to nonconcerous tissue. For this purpose, matchednoncancerous tissue (i.e. “normal” tissue) is of limited availability. A“universal” epithelial control was prepared by pooling noncanceroustissues of epithelial origin, including liver, kidney, and lung. RNAisolated from the pooled tissue represents a mixture of expressed geneproducts from these tissues. The pooled control referred to hereinafteras the “control” sample, was an effective control for relative geneexpression studies of tumor tissue and tumorigenic cell lines.Microarray hybridization experiments using the pooled control samplesgenerated a linear plot in a 2-color analysis as disclosed herein.Because the test and control samples have many genes expressed atsimilar quantitative levels, a plot of intensity data for all of thetarget molecules that formed complexes with the control and test probesyielded a linear clustering of the data. The slope of the line fitted tothese data in a 2-color analysis was then used to normalize the ratiosof test to control within each experiment. The normalized ratios fromvarious experiments were then compared and used to identify clusteringof gene expression, and genes differentially expressed in diseasedtissue versus normal tissue across many different tissue samples. Thus,the pooled “universal” control sample not only allowed effectiverelative gene expression determinations in a simple 2-sample comparison,it also allowed multi-sample comparisons across several experiments.

Example 3 Microarray Slide Preparation

Activated glass slides used for attachment of target moleculepolynucleotides in nucleic acid microarray preparation are commonlytreated with polylysine (see, for example, U.S. Pat. No. 5,807,522) ororganosilane (See, for example, WO 01/06011; WO 00/40593; U.S. Pat. No.5,760,130; and Weiler et al., Nucleic Acids Research 25(14):2792-2799(1997)). For the purposes of the present invention, organosilane-basedtreatment of the glass slide was preferred because it allowed specificnucleic acid sequence end attachment via a covalently attached primaryamine on the nucleic acid as disclosed herein. Such specific attachmentis advantageous for specific positioning of nucleic acid sequences on amicroarray slide, thereby ensuring attachment of the nucleic acid whilerendering it free to hybridize efficiently with complementary sequencesin a probe.

It was discovered as part of the present invention that even unmodifiednucleic acids (such as target DNA) can attach to a glass slide treatedwith 3-aminopropyltriethoxysilane (APS) followed by attachment ofphenylene diisothiocyanate, suggesting that the nucleic acids may alsobe attaching a functional group on unmodified DNA (for example, at the5′ end of an unmodified promer used to amplify nucleic acids forarraying by PCR, or amines on unmodified DNA bases). Thus, the inventioninvolves the attachment of unmodified polynucleotides to an activatedmicroarray slide of the invention.

The present inventors also discovered that the solvent used for silanetreatment of glass slides has a marked affect on the fluorescentbackground observed in microarray analysis. Acetone, the commonly usedsolvent for dissolving silane during glass slide treatment, caused ahigh and/or non-uniform fluorescent background during imaging. Methanolis occasionally used as a solvent for silanization (see, for example,<http://sgio2.biotec.psu.edu/protocols/silanize.html> (last visited Mar.13, 2001). Methanol is disadvantageous because water present in methanolquenches the silanizing reaction and limits efficient coating of the aglass microarray slide. For examples of other procedures forsilanization in solvents other than toluene, see, WO 01/06011; WO00/40593; U.S. Pat. No. 5,760,130; and Weiler et al., (1997), supra).Because efficient silanization and low background is preferred formaximum signal-to-noise ratio and highest sensitivity, an alternativesolvent was sought. Toluene was found to be a superior solvent forsilane treatment because longer glass treatment could be used to ensureoptimal coating while avoiding high fluorescent background. Acetone isstill useful in the glass slide treatment method of the invention, butits use is preferably confined to drying steps where contact withacetone is of relatively short duration.

Preparation of Activated Microarray Slides:

According to the method of the invention, glass slides were treatedusing the following protocol to prepare them for use in making nucleicacid microarray slides.

Cleaning Glass Slides: Glass microscope slides (standard size) were usedfor the present experiment. Throughout the procedure, the slides werehandled with solvent-proof gloved hands. Thirty slides were loaded ontoa clean metal rack and the rack was lowered in a clean ultrasoniccleaner chamber filled with 1% Liquinox™ (Alconox, NY, N.Y.) in highlypurified water, such as by reverse osmosis (designated “SQ water”;MilliQ™ System, Millipore Corp., Bedford, Mass.) The Liquinox solutionwas heated to approximately 50 C in the ultrasonic cleaner prior toimmersing the slides. The slides were cleaned ultrasonically for 30 min.at 50 C. The same solution of Liquinox may be used to cleanapproximately 4 batches of 30 slides per batch. After cleaning, theslides were transferred to a plastic container filled with deionizedwater. The plastic container is preferably used only for rinsing cleanedslides to avoid contamination of the slides with extraneous material.The slides were rinsed three times with running deionized water and thenplaced on a shaker. The rinsing and shaking steps were repeated sixtimes to ensure thorough rinsing. The final rinse was with SQ water. Theslides were stored in SQ water until use.

In a preferred cleaning method according to the invention, slides wereloaded in glass racks, 20 slides per rack, and cleaned in a cleanultrasonic cleaner chamber filled with 3%, GLPC-Acid™ in highly purifiedwater, such as by reverse osmosis (designated “SQ water,” MilliQ™System, Millipore Corp. Bedford, Mass.), for 20 minutes at 65° C. Aftercleaning, the slides were rinsed thoroughly with deionized water. Theslides were then placed in an ultrasonic cleaner chamber containing 0.5%sodium hydroxide, 50% ethanol and treated for 10 minutes at 65° C. Theslides were rinsed very thoroughly with deionized water and the finalrinse was with SQ water. The slides were stored in SQ water until usethe next day.

All subsequent procedures for slide silanization were performed in awell-ventilated fume hood.

Drying Slides: The clean slides were transferred in the metal rack to aglass chamber. The slides were covered with acetone, shaken briefly, andremoved from the acetone. The slides are allowed to drain and then dryin the fume hood. The slides were protected from exposure to dusty airthat may be drawn into the fume hood by placing the slides behind theglass chamber in the hood and/or placing them back in the glass chamberafter the acetone is removed and the chamber allowed to dry. The slidesremained in the glass chamber until dry and free of water or acetonebecause these solvents are problematic: water interferes withsilanization and acetone causes high fluorescent background.

Silanizing Glass Slides: Screw-cap Coplin staining jars were cleaned anddried completely. Preferably drying is performed in a drying oven. Theclean glass slides were transferred into the dry staining jars usinggloved hands and forceps by handling the slides only at the corners. Asolution of 10% 3-aminopropyltriethoxysilane in toluene (substantiallywater-free as purchased from Burdick and Jackson) was prepared by addingthe silane to the toluene and swirling to mix. Immediately after mixing,the silane solution was poured over the slides in each jar.Approximately 550 ml silane solution filled 6 jars. The jars werequickly covered with the screw-caps such that air and moisture wereexcluded from each jar to avoid precipitation of silane polymers on theslides. The slides were silanized overnight.

In a preferred method of silanizing glass slides according to theinvention, a solution of 2% 3-aminopropyltriethoxysilane in toluene(reagent grade) was prepared by adding the silane to the toluene andswirling to mix. Immediately after mixing, the silane solution waspoured over the slides in each glass chamber. The glass chambers werecompletely filled to the top edge and a lid was placed on top. Theslides were silanized 1-4 hours at room temperature. Preferably, the 2%silanizing procedure is applied to glass slides cleaned in 0.5% NaOH, 5%ethanol (as disclosed supra).

Washing Silanized Slides: Following silanization, slides were washed bythe following procedure. Glass washing chambers containing slide rackswere filled with toluene. A glass chamber that holds 10 slides is filledwith 250 ml toluene and a chamber that holds 20 slides requiresapproximately 400 ml toluene. The silanized slides were transferred fromthe silanization solution to racks submerged in toluene in the glasschambers using forceps to handle the slides only at the corners andwithout allowing the slides to dry during the transfer. In anotherwashing procedure and at the end of the silanization period, thesilanization chamber was emptied and filled with toluene such that therack of slides was covered.

At this point in either of these washing procedures, a clean glass lidwas placed on top of the chamber and the chamber was agitated for 2-6min. The glass lid was then removed and inverted on the counter top toprovide a platform on which the rack of washed slides were placed. Thetoluene was discarded from the chamber. The toluene wash was repeatedtwice. The slides were not allowed to dry during the wash procedures.

The third toluene wash was discarded, the chamber drained, and methanolwas added to the chamber. Slides were submerged in the methanol andagitated for approximately 5 min. Slides were washed twice withagitation in SQ water for 5 min per wash. The slides were then washedtwice in methanol for approximately 4-5 min. with agitation.

As the final wash step, the slides were rinsed with acetone for 1 min tospeed drying. The slides were allowed to dry completely in the fumehood. The slides were protected from dust by placing them in the emptyglass chambers used for the wash steps. At this stage, the slides werestable for approximately one hour. In another method following theacetone rinse, slides were rinsed in dimethylformamide (DMF). The DMFwas then drained from the chamber. After these wash procedures, theslides were prepared for attachment of a bifunctional linker reagent.

PDITC Attachment to Silanized Slides: The surface of the silanizedslides was next cross-linked using 1,4-phenylene diisothiocyanate(PDITC), a bifunctional cross-linking agent (see Greg T. Hermanson,Bioconjugate Techniques, Academic Press (1996)) capable of reacting withsilane on the glass slide at one end, and with amino-derivatizedmicroarray DNA at the other end. Microarray DNA is thus firmly attachedto the glass surface. The PDITC linkage is water sensitive, however. Asa result, the slides must remain free of water until after attachment ofthe target molecule, such as a target polynucleotide.

The PDITC solution was prepared as follows. To a solution of 90%dimethyl formamide (DMF) and 10% pyridine, an appropriate amount ofsolid PDITC was added to provide a 0.20-0.25% PDITC concentration and,as expected, the solution was yellow. Due its reactivity, solid PDITCwas handled quickly and stored under argon.

The PDITC solution was poured over the silanized slides, still in theglass chambers in the fume hood, and the chambers were completelyfilled. The glass lids were placed on the chambers and each chamber wascovered with foil to block exposure to light. The slides were incubatedin PDITC for 2 hours.

Following incubation, the PDITC solution was removed. DMF was added tothe chambers and the slides were agitated for 3-5 min. The DMF wash wasrepeated twice more with agitation for approximately 5 min per wash.

The slides were then washed twice with methanol for 3-5 min. withagitation. The slides were not left in methanol for longer than 5 min.because traces of water in methanol could react with the PDITC. Theslides were washed 3 times with agitation in acetone for 3-5 min. perwash. The slides were then dried completely in the fume hood, protectedfrom dust. The PDITC-treated slides were then stored in a dry cabinet.The slides are stable under these conditions for at least 3 months.

Example 4 Attaching Target Molecules to an Activated Microarray Slide.

It is understood that microarrays may be prepared by the user orpurchased commercially. Descriptions of microarrays on glass slides areavailable in, for example, U.S. Pat. No. 6,040,138. Generally, a DNAmicroarray on a glass slide contains at least 100, preferably at least400 or more DNA samples or at least partially known sequence in knownlocations on the slide at a density of at least 60 oligonucleotidesequences per square centimeter. The microarray sequences may beoligonucleotides of 5-100 nucleotides in length, or the sequences may bepolynucleotides from 50 nt to 10 kb in length, or they may be fulllength gene sequences. A sufficient portion of each sequence must beknown so that it is distinguishable from the other sequences, and itmust be long enough to hybridize to a labeled probe under the conditionsused.

Preparation of Target Nucleic Acid Sequences:

In this example, nucleic acid sequences of interest (“target sequences,”“target polynucleotides,” or “targets”) were generated from full lengthor partial cDNA clones. Optionally, a target was cloned into a vectorfor ease of manipulation. The target sequence (i.e. non-vector nucleicacid sequence of interest) was amplified by the polymerase chainreaction (PCR) using “Klentaq GC melt” DNA polymerase (Clontech). Thisenzyme provided a high success rate of amplifying DNA inserts, withuniform yields, across a range of templates that varied in both length(0.25-4 kb) and nucleotide composition.

As disclosed herein, an unmodified polynucleotide attaches directly toan activated glass slide prepared by silanizing with an organosilane intoluene, followed by reaction with a multifunctional linker reagent thatis capable of reacting with the unmodified polynucleotide. As theexamples herein disclose, the organosilane may be APS and themultifunctional linker reagent may be PDITC.

Alternatively, the target molecule may be modified by incorporation of areactive group in the target molecule, which reactive group is reactivewith a functionality on the multifunctional linker reagent of theactivated microarray slide of the invention. According to thisalternative method of the invention and simultaneous with amplificationof target sequences, the amplified targets were modified to comprise alinker for covalent attachment to a solid support of a microarray. Toaccomplish simultaneous amplification and modification, PCR primers hadat least two features. First, the PCR primers were complementary to thevector sequences into which the target DNA was inserted, therebyensuring amplification of the complete target sequence. Further, theprimer from which the modified single strand target DNA would begenerated comprised a reactive moiety: a primary amine linked to theprimer's 5′ end via a linker, preferably an alkyl linker, such as a—(CH₂)₆— linker. For the purpose of this example, such a primer had thefollowing general structure: 5′ NH₂—(CH₂)₆-dNx 3′, where NH₂ is aprimary amine group, (CH₂)₆ is a methylene linker, and dNx is anucleotide sequence, preferably an oligonucleotide sequence (DNA in thisexample), complementary to a portion of the vector into which the targetDNA was inserted (primers were synthesized at Genentech, Inc. So. SanFrancisco, Calif., USA). Preferably, the dNx sequence hybridizes to avector sequence near the target insert such that enzyme-drivenelongation of the primer into the target sequence using twovector-specific primers that flank the target sequences. Nucleic acidsynthesis resulted in formation of a double stranded nucleic acidsequence complementary to the target sequence, wherein the complementaryregion is at least 10 nucleotide bases is length. Thus, following PCRamplification, each target sequence comprised a primary amino group onits 5′ end, which amino group was capable of reacting with a reactivegroup on an activated slide. For example, as disclosed herein by anon-limiting example, a primary amine incorporated into a polynucleotideallows immobilization of the polynucleotide on an activated glass slide.According to the invention, a glass slide is activated by silanizing intoluene with a organosilane that is then reacted with a multifunctionallinker reagent. The multifunctional linker reagent is reactive with boththe organosilane on the surface of the glass and with a primary amine ofa modified polynucleotide as disclosed above.

Prior to immobilization on an activated slide, PCR-amplified doublestranded target DNA sequences were purified using glass-fiber filters(Qiagen, Valencia, Calif.). A portion of the purified sequences wasanalyzed by agarose gel electrophoresis for correct molecular weight,purity (e.g. a single band representing a single product and not amixture of clones or genes) and approximate yield or DNA (estimated byfluorescent staining with ethidium bromide following standardprocedures).

The primary amine-modified target sequences were resuspended in anarraying buffer (500 mM sodium chloride, 100 mM sodium borate, pH 9.3,which promotes reaction between the primary amine of the modified targetDNA and the PDITC-derivatized, silanized glass surface, resulting incovalent attachment of the target DNA to the glass slide. The slideswere ready for use according to the invention, increased attachment andimproved detection intensity was acheived when the slides were allowedto remain at ambient temperature and humidity in the dark overnight,such as for approximately 10-16 hours. A concentration of modifiedtarget sequence of at least 0.1 μg/μl provided successful covalentattachment to the activated glass slides, good spot morphology, and asufficient number of covalently attached target sequences such that theywere in excess relative to fluorescently labeled cDNA probes appliedduring subsequent hybridization reactions. This permitted quantitativemeasurement of the absolute fluorescent signals obtained after probehybridization.

In this example, a two-step protocol was used to attach nucleic acids,such as gene sequences, to the silanized, PDITC-treated glass slidesprepared according to the present invention. It is understood that fewersteps or more steps may be used as long as any silanizing step isperformed in toluene in the absence of acetone or an an alcoholaccording to the present invention.

As disclosed, supra, the slides were first silanized with3-aminoproplytriethoxysilane (APS) in toluene. The slides were thentreated with PDITC (1,4-phenylene diisothiocyanate), a multifunctionallinker reagent which contains two amine-reactive isothiocyanate groups.One of the isothiocyanate groups reacts with the amine group of theorganosilane. The second isothiocyanate group is available to react witha primary amine present on the 5′ ends of the modified target DNA (seeExample 1), thereby providing the means of attaching the target DNA tothe glass slide during spotting of the DNA onto the microarray. Afterattaching the modified target sequences, the slides were washed once inwater containing 0.2% SDS, then washed three times in SQ water, andfinally dipped in ethanol and dried. Slides cleaned, silanized, andPDITC-treated according to the method of the invention were superiorsubstrates for nucleic acid microarrays because fluorescent backgroundwas minimized, and hybridization was enhanced by minimizingover-attachment of the arrayed target DNA, thereby providing asurprising increase in detection levels over previous methods.

Microarrays Comprising Single Stranded Target Oligonucleotides

Improved microarrays comprising single stranded target oligonucleotidesare encompassed by the present invention. A non-limiting example of thearrays and a method of making them follows.

Single stranded target DNA for array fabrication was synthesized bystandard solid-phase methods with a 3′-C7 amino linker (Glenn Research,Sterling, Va.) with or without hexethyleneglycol spacers (S18) (GlennResearch, Sterling, Va.) incorporated between the 3′-end of thesynthetic DNA and the C7 linker.

Single stranded DNA molecules, such as chemically synthesized targetoligonucleotides of approximately 50 to 100 nucleotides in length wereimmobilized onto activated microarray slides of the invention (e.g.aminosilane in toluene/PDITC-treated glass) by standard microarrayprinting techniques. The printing solution comprised oligonucleotides ata concentration of up to 10 μM in 0.1 M borate, 0.5 M NaCl, pH 9.3. Theslides were dried overnight at 20° C. and ambient room humidity. It wasdiscovered as part of the present invention that drying overnightgenerated microarrays capable of providing an increased fluorescentsignal when hybridized with polynucleotide probes of the invention.

Improved detection signal was demonstrated by hybridizing acomplementary fluorescein-labeled 100 mer single strand DNA fragment tothe single stranded target oligonucleotide DNA arrays as disclosed,supra, revealed that hybridization signal intensity was dependent onimmobilized DNA length, with longer DNA strands providing a strongersignal. In addition, varying the number of S18 repeats from 0 to 6revealed increasing signal intensity with increasing tether length. Thecombination of a 100 nucleotide single stranded target DNA molecule with6-S18 repeats and a C7 amino linker provided highest hybridizationsignal intensity. Accordingly, microarrays of the invention comprisingsingle stranded target DNA oligonucleotides are improved when thedistance of the oligonucleotide from the solid surface and DNA chainlength are increased.

Spotting Target Molecules onto Activated Slide

Target DNA (modified or unmodified) in 5-10 μl 100 mM sodium borate pH9.3, 500 mM sodium chloride, in 384 well plates, was used for arrayingthe target DNA onto activated microarray slides of the invention.Arraying, (also termed printing or spotting) target molecules on anarray slide, was performed using an automated microarraying deviceequipped with a printing pin having a 80 micron internal width (TeleChemInternational. Inc., model no. CMP2, “Chipmaker 2 Microspotting Pins”).Approximately 0.5-1 nl of target solution was deposited at each arrayelement (spot or location) using the printing pin. Spot size wasregulated at 100-140 microns in diameter due to the tip diameter and thenature of the surface generated on the slides prepared according to theinvention. Due to the buffer used for printing and the reactivity of theslides of the invention, nucleic acid molecules attach rapidly with nofurther manipulations. It was discovered as part of the invention thatleaving the printed slides at ambient conditions overnight increasedattachment of target DNA to the microarray slides in some cases.

Following spotting, slides were placed in glass racks and washed in 0.2%SDS, followed by three washes in SQ water, followed by an ethanol rinse.This washing procedure removes unattached target DNA and modifiesunreacted thiocyanate functionalities. Printed, washed slides wereallowed to dry and stored in slide boxes in the dark under ambientconditions.

Example 5 Hybridization Method for Microarray Analysis

The microarray hybridization method disclosed herein allows enhancednucleic interaction for improved hybridization and highersignal-to-noise ratio for more sensitive detection. Greater sensitivityis useful when samples, such as tissue samples, are small and limited.

According to the present invention, formamide and/or dimethylsulfoxideare used to suspend labeled oligonucleotide probes because thefluorescently labeled DNA probe is more soluble in these polar ogranicsolvents. Preferably, the amount of polar organic solvent in thehybridization solution is not more than 50%, 40%, 30%, 25%, or 20%.According to the invention, the proportion of DMSO is from 0% to andincluding 50%, from 0 to and including 40%, from 0 to and including 30%,from 0 to and including 25%, and from 0 to and including 20%. Similarly,the proportion of formamide is from 0% to and including 50%, from 0 toand including 40%, from 0 to and including 30%, from 0 to and including25%, and from 0 to and including 20%. Thus, according to the invention,the total amount of polar organic solvent (either DMSO or formamide)does not exceed 50%, for example, which the relative proportion of DMSOto formamide is varied from such that the sum of the proportions ofthese organic solvents does not exceed 50%, in this example.

In addition, it was discovered by the present inventors that theomission of detergent, sodium dodecyl sulfate (SDS) for example, fromthe hybridization conditions improved detection. It was discovered thatSDS caused the formation of colloidal; complexes with the fluoorescentlylabeled DNA probe, causing the probes to precipitate out of solution,limiting detection, and/or causing unwanted detection variability,and/or very high non-specific fluorescent background. The absence of thesolid surface wetting capabilities of SDS were overcome by the use offormamide in the hybridization and the glass surface treatment disclosedherein.

The microarray hybridization method of the invention comprises thefollowing protocol. Before application of the probe, the microarray wasdenatured by placing it at 95° C. for 2 min. The microarray was thensubmerged in cold ethanol (approximately 20° C.) to quickly cool it toroom temperature and to maintain the denatured state of the sequences inthe array. Probes were resuspended in a final concentration of 5×SSC.50% formamide. The resuspension was allowed to continue for at least 3hours and up to overnight (e.g. approximately 10-16 hours in the dark.The control and test probes were pooled, heated to 95-100° C. for 45seconds, and, while hot, applied as 10 μl aliquots to the surface or thedenatured microarray slide, which was on a slide warmer at approximately50° C. Following application of the probes, a clean glass coverslip wascarefully placed over the array to cover it. The covered microarrayslide was placed in a hybridization chamber at 37° C. overnight. Thehybridization chamber may be any vapor-tight, chemically inertcontainer. For example, the hybridization chamber used in the presentexample was a plastic container having a vapor-tight plastic lid intowhich were placed absorbent material, such as paper towels, wet with50:50 formamide:water. The interior of the chamber was allowed toequilibrate at 37 C for at least 30 min prior to use.

Hybridization in Alkylammonium Salt, DMSO, and Formamide

It was discovered as part of the invention that alkylammonium salts,dimethylsulfoxide (DMSO), and formamide in the microarray hybridizationbuffer improved detection sensitivity.

Alexa-dye (Molecular Probes, Eugene, Oreg.) labelled cDNA probes,either + or − strand, may be hybridized to cDNA or oligonucleotidearrays in 2.4 M TEACl (Alfa Aesar, Ward Hill, Mass.) or 3.0 M TEACl(Sigma. St Louis, Mo.) with 50 mM Tris (Sigma). 2 mM EDTA (Sigma) at pH8.0. The polar solvents formamide (Life Technologies, Rockville, Md.)and dimethylsulfoxide (DMSO) (Sigma) were also included in the arrayhybridization solution in varying proportions up to a final totalconcentration of DMSO and formamide of 25% (v/v). In other words,formamide and DMSO concentrations may vary from 25% (v/v) formamide and0% (v/v) DMSO to 0% (v/v) formamide and 25% (v/v) DMSO, for example 20%(v/v) formamide, 5% (v/v) DMSO. It was found as part of the presentinvention that hybridization of a fluorescently labeled polynucleotideto an oligonucleotide array as disclosed herein was improved when TEACland DMSO were in the hybridization buffer.

For example, signal intensity using a first hybridization buffer(Buffer 1) comprising 50% (v/v) formamide, 5×SSC buffer was compared toa second hybridization buffer (Buffer 2) comprising 2.4 M TEACl, 50 mMTris, 2 mM EDTA, pH 8.0, with 20% formamide/5% (v/v) DMSO. Separate (−)strand labeled cDNA probe mixture were prepared with Alexa488-dUTP orAlexa546-dUTP (Molecular Probes, Eugene, Oreg.) by second strandsynthesis with simultaneous label incorporation as disclosed herein.Each labeled probe mixture was divided into equal aliquots and vacuumdried. Samples were resuspended in either 50% (v/v) formamide, 5×SSCbuffer (Buffer 1) or 2.4 M TEACl, 50 mM Tris, 2 mM EDTA, pH 8.0, with20% formamide/5% (v/v) DMSO (Buffer 2) 488 and 456 labeled probes werepooled and each different probe pool was hybridized to one of amicroarray duplicate. The results demonstrated fluorescence signalintensity was improved for each label in Buffer 2 relative to Buffer 1as a result of the addition of TEACl and DMSO. The hybridization signalfound with 2.4 M TEACl, 50 mM Tris, 2 mM EDTA, pH 8.0, with 20%formamide/5% (v/v) DMSO was increased 3-5 fold over the signal obtainedin hybridization buffer lacking TEACl and DMSO.

After hybridization, the microarray slides were taken from the chamber.The coverslip was carefully removed and the slides were washed in 2×SSC,0.2% SDS for 2-5 min. followed by a wash with 0.2×SSC, 0.2% SDS for 2-5minutes. The slide was covered with a new, clean coverslip to keep thearray region wet with the last wash solution while imaging of thehybridized array was performed. Imaging the slides while wet avoidsquenching of the chromophores, thus improving both the absolute signaland the quantitative nature of the signal. The top and bottom of theslide were otherwise kept dry. Imaging did not bleach the chromophoresand the hybridized microarrays may be stored in the dark for re-imagingfor at least 60 days.

Example 6 Detection Method

Means for detecting the labeled hybridized probes are well known tothose skilled in the art. In the present example where fluroescentlylabeled probes were applied to densely arrayed nucleic acid sequences,detection is preferably performed by fluorescence imaging.Alternatively, a CCD camera imaging system was used. For, example,excitation of the chromophores using fluorescence spectroscopy occurs byexposing the hybridized slide to a fluorescent laser or other lightsource through a filter specific for the desired excitation wavelength.Fluorescent emission was detected at the discrete emission wavelengthfor each chromophore. The relative emission of test and control probeswas analyzed according to the chromophore incorporated into each probetype and the specific microarray member to which a probe hybridized. Theanalysis provided quantitative information on the relative expression ofthe genes in diseased tissue. Where automated detection and analysis aredesired, an automated system for detecting and quantifying relativehybridization is found, for example, in U.S. Pat. No. 5,143,854, whichdetection procedures are herein incorporated by reference.

Microarray slides hybridized with a mixture of test and control probeswere viewed using an imaging device configured for fluorescenceexcitation at 488 nm and 546 nm and detection at the appropriatecorresponding wavelengths (e.g. 530 nm and 590 nm, respectively). Thedevice was an imaging fluorimeter that produces a two-dimensionalelectronic image of emission intensities of the array spots. A deviceuseful for such detection is, for example, an ArrayWoRx™ microarrayscanner (Applied Precision, Inc., Issaquah, Wash., USA). A detaileddescription of the detection process is available from the supplier(see, for example, <www.appliedprecision.com>, last visited Mar. 23,2000). Briefly, white light is directed through an excitation filter todeliver selected monochromatic light onto to the hybridized sample.Fluorescent emission is focused on a CCD camera having high resolutioncapability. The collected detection data may be concurrently orsubsequently analyzed and reported. Preferably, each emission color isrepresented separately for display purposes.

Alternative devices and procedures known in the art are useful for thedetection and analysis of the relative complex formation of control andtest probes with target polynucleotides according to the invention.Other useful procedures are found in, for example, WO 00/32824(published Jun. 8, 2000), WO 00/04188 (published Jan. 27, 2000).

FIGS. 1-4 are examples of microarray experiment results, where themicroarrays were prepared and treated according to the methods of theinvention disclosed herein (i.e., RNA purification, slide preparation,probe synthesis, and probe hybridization). FIG. 1 is a photograph ofmicroarrays hybridized with probes synthesized from a very smallquantity of tumor cells microdissected from tumor tissue. Thesignal-to-noise is high allowing improved detection of hybridizedprobes. FIGS. 2A and 2B indicate that detection is comparable for probessynthesized from paraffin-embedded liver versus fresh, frozen liver.FIGS. 2C and 2D demonstrate detection of gene expression in fresh frozenversus paraffin-embedded colon tissue from the same patient. The linearclustering of the detection data from the two differently preservedtissue samples shown in the scatter plot of FIG. 2D illustrates thequantitative gene expression obtained from fresh-frozen versusformalin-fixed, paraffin-embedded tissue is very similar. FIGS. 3A and3B show a comparison of gene expression in colon tumor relative to geneexpression in the control tissue comprising pooled epithilial tissue.Emission intensity of each spot at the emission wavelengths of thechromophores are compared and analyzed to determine the actual relativegene expression in diseased and healthy tissue. FIGS. 4A-4C show thatwhere RNA starting material from an ovarian carcinoma cell line waslimited, detection of the probes hybridized to the array was possiblefor sDNA probes synthesized from 200 pg (FIG. 4A), 20 pg (FIG. 4B), and2 pg (FIG. 4C) with only one round of amplification by cRNA reversetransciption to labeled sDNA in a 5-hour reaction, as disclosed herein.A 1-color analysis of fluorescence intensity is shown.

The foregoing written specification is considered sufficient to enableone skilled in the art to practice the invention. The present inventionis not to be limited in scope by the examples provided since theembodiments are intended as illustrative of certain aspects of theinvention and any embodiments that are functionally equivalent arewithin the scope of the this invention. The presentation of examplesherein does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice or any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustrations that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. The disclosures of allcitations in the specification are expressly incorporated herein byreference.

1. A microarray comprising a surface silanized with a silane in toluenein the absence of acetone or an alcohol, and a target molecule, whereinthe target molecule is attached to the surface via the silane.
 2. Amicroarray comprising a surface silanized with a silane in toluene inthe absence of acetone or an alcohol, a linker, and a target molecule,wherein the target molecule is attached to the surface via the linker.3. The microarray of claim 2, wherein the target molecule is apolynucleotide.
 4. The microarray of claim 3, wherein the polynucleotideis selected from a group consisting of an oligonucleotide, DNA,amplified DNA, cDNA, single stranded DNA, double stranded DNA, PNA, RNA,and mRNA.
 5. The microarray of claim 4, wherein the polynucleotide has alength in the range of about 3 bp to 10 kb.
 6. The microarray of claim5, wherein the length is in the range of about 100 bp to 5 kb.
 7. Themicroarray of claim 6, wherein the length is in the range of about 0.3kb to 3 kb.
 8. The microarray of claim 7, wherein the length is in therange of about 0.5 kb to 2 kb.
 9. The microarray of claim 4, wherein thepolynucleotide is an oligonucleotide and the oligonucleotide is 25-1000bp, 25-500, 30-200, and 50-100 bp in length.
 10. The microarray of claim2, wherein the target molecule is a polynucleotide and comprises anamine.
 11. The microarray of claim 10, wherein the amine group is aprimary amine.
 12. The microarray of claim 11, wherein the primary amineis at the 5′ end of the polynucleotide.
 13. The microarray of claim 11,wherein the primary amine is attached at the 5′ end of thepolynucleotide via a linker, wherein the linker comprises one or moremonomers of 1-20 carbon atoms, and wherein the monomer comprises alinear chain of carbons or a ring or both.
 14. The microarray of claim12, wherein the polynucleotide is prepared by extending a nucleic acidprimer comprising a primary amine at its 5′ end.
 15. The microarray ofclaim 2, wherein the substrate surface is selected from the groupconsisting of polymeric materials, glasses, ceramics, natural fibers,nylon, nitrocellulose, silicons, metals, and composites thereof.
 16. Themicroarray of claim 15, wherein the substrate surface is planar.
 17. Themicroarray of claim 15, wherein the substrate is in a form or threads,sheets, films, gels, membranes, beads, plates, and like structures. 18.The microarray of claim 15, wherein the substrate surface is glass. 19.The microarray of claim 18, wherein the substrate is a glass slide. 20.The microarray of claim 2, wherein the target molecule is attached aftercontacting the target molecule with the surface by a technique selectedfrom the group consisting of printing, capillary device contactprinting, microfluidic channel printing, deposition on a mask, andelectrochemical-based printing.
 21. The microarray of claim 20, whereinthe target molecule is unmodified prior to the contacting.
 22. Themicroarray of claim 21, wherein the target molecule is modified tocomprise an amine prior to the contacting.
 23. The microarray of claim22, wherein the amine is a primary amine.
 24. The microarray of claim23, wherein the target molecule is a polynucleotide and the primaryamine is at the 5′ end of the polynucleotide.
 25. A microarray preparedby a method comprising: (a) providing a multifunctional linker reagentcomprising two or more reactive groups capable of reacting with afunctional group on a surface of a microarray substrate and capable ofreacting with a target molecule; (b) activating the substrate surfacefor immobilizing the target molecule, by silanizing the surface with asilane in toluene in the absence of acetone or an alcohol, wherein thesilane comprises a functionality reactive with the multifunctionallinker reagent, and wherein the activating further comprisesimmobilizing the multi functional linker reagent on the silanizedsurface by attaching the multifunctional linker reagent to the silanevia a first reactive group of the linker reagent and a reactive group ofthe silane; (c) providing a solution comprising a target molecule havingone or more functional groups reactive with a second reactive group ofthe immobilized multifunctional linker reagent; (d) attaching the targetmolecule to the substrate surface by contacting the target molecule withthe activated substrate surface under conditions that promote attachmentof the target molecule to the immobilized multifunctional linkerreagent.
 26. The microarray of claim 25, wherein the target molecule isa polynucleotide, and wherein the contacting of step (d) is carried outby spotting the polynucleotide on an activated substrate surface. 27.The microarray of claim 26, wherein the polynucleotide is unmodified.28. The microarray of claim 26, wherein the polynucleotide is modifiedwith an amine group.
 29. The microarray of claim 28, wherein the aminegroup is a primary amine at the 5′ end of the polynucleotide.
 30. Themicroarray of claim 26, wherein the polynucleotide is spotted on thesurface at a concentration in the range of approximately 0.1 μg/μl toand including approximately 3 μg/μl.
 31. The microarray of claim 25,wherein the attaching of step (d) occurs in a pH range from pH 6 to andincluding pH
 10. 32. The microarray of claim 31, wherein the pH range isfrom pH 6.5 to and including pH 9.7.
 33. The microarray of claim 32,wherein the pH range is from pH 7 to and including pH 9.4.
 34. Themicroarray of claim 33, wherein the pH is 9.3.
 35. The microarray ofclaim 25, wherein the attaching is allowed to occur for a time periodfrom 1 minute to and including 24 hours.
 36. The microarray of claim 35,wherein the time period is from 1-24 hours.
 37. The microarray of claim36, wherein the time period is from 5-18 hours.
 38. The microarray ofclaim 37, wherein the time period is from 10-16 hours.
 39. Themicroarray of claim 38, wherein the time period is from 12-14 hours. 40.The microarray of claim 25, wherein the method of preparing themicroarray further comprises, after step (d), blocking unreactedreactive groups.
 41. An activated slide comprising a substrate surfacecomprising a silane attached thereto, wherein the silanizing was intoluene, in the absence of acetone or an alcohol, and wherein theattached silane comprises at least one reactive functionality that iscapable of reacting with a compound to immobilize the compound on thesubstrate surface.
 42. The activated slide of claim 41, wherein thecompound is selected from the group consisting of a modified targetmolecule, an unmodified target molecule, and a multifunctional linkerreagent.
 43. The activated slide of claim 42, wherein the compound is amultifunctional linker reagent comprising at least one reactive groupcapable of reacting with a target molecule to immobilize the targetmolecule on the substrate.
 44. The activated slide of claim 42, whereinthe target molecule is an unmodified polynucleotide comprising a nativereactive group capable of reacting with the reactive functionality ofthe silane.
 45. The activated slide of claim 43, wherein the targetmolecule is an unmodified polynucleotide comprising a native reactivegroup capable of reacting with the reactive group of the multifunctionallinker reagent.
 46. The activated slide of claim 43, wherein the targetmolecule is a modified polynucleotide comprising a non-native reactivegroup capable of reacting with the reactive group of the multifunctionallinker reagent.
 47. The activated slide of claim 46, wherein the targetmolecule is a polynucleotide and the non-native reactive group is anamine.
 48. The activated slide of claim 47, wherein the amine is aprimary amine.
 49. The activated slide of claim 48, wherein the primaryamine is at the 5′ end of the polynucleotide.
 50. The activated slide ofclaim 41, wherein the silane is an alkyl silane and the alkyl moiety isselected from the group consisting of an ethyl-, a propyl-, a butyl-, apentyl-, a hexyl-, a heptyl-, an octyl-, a nonyl-, and a decylalkylmoiety, and the reactive functionality of the silane is selected fromthe group consisting of an amine, a hydroxyl moiety, an epoxide, athiol, and a halide, and the reactive functionality is covalently linkedto the alkyl moiety.
 51. The activated slide of claim 50, wherein thereactive functionality of the silane is a primary amine on the alkylmoiety, and wherein at least one reactive group of the multifunctionallinker reagent is a thiocyanate moiety, and wherein the multifunctionallinker reagent is immobilized by covalent reaction with the primaryamine of the silane of the silanized surface.
 52. A method of activatinga glass slide for immobilizing a target molecule, the method comprisingsilanizing the slide with a silane in toluene in the absence of acetoneor an alcohol, wherein the silane is an alkyl silane and the alkylmoiety is selected from the group consisting of an ethyl-, a propyl-, abutyl-, a pentyl-, a hexyl-, a heptyl-, an octyl-, a nonyl-, and adecylalkyl moiety, and the reactive functionality of the silane isselected from the group consisting of an amine, a hydroxyl moiety, anepoxide, a thiol, and a halide, and the reactive functionality iscovalently linked to the alkyl moiety.
 53. The method of claim 52further comprising reacting the silane with a multifunctional linkerreagent comprising at least one reactive group capable of reacting withthe silane and at least one reactive group capable of reacting with thetarget molecule for immobilizing the target molecule, wherein thereactive functionality of the silane is a primary amine on the alkylmoiety, and wherein at least one reactive group of the multifunctionallinker reagent is a thiocyanate moiety, and wherein the multifunctionallinker reagent is immobilized by covalent reaction with the primaryamine of the silane of the silanized surface.
 54. The method or claim52, wherein the silane is an alkyl silane and the alkyl moiety isselected from the group consisting of an ethyl-, a propyl-, a butyl-, apentyl-, a hexyl-, a heptyl-, an octyl-, a nonyl-, and a decylalkylmoiety, and the reactive functionality of the silane is selected fromthe group consisting of an amine, a hydroxyl moiety, an epoxide, athiol, and a halide, and the reactive functionality is covalently linkedto the alkyl moiety.
 55. A method of preparing a microarray, the methodcomprising: (a) providing an activated slide comprising a substratesurface comprising a silane attached thereto, wherein the silanizing wasin toluene, in the absence of acetone or an alcohol, and wherein theattached silane comprises at least one reactive functionality that iscapable of reacting to immobilize a target molecule on the substratesurface; (b) reacting the activated slide surface with the targetmolecule under conditions to immobilize the target molecule, wherein thetarget molecule is selected from the group consisting of a nucleic acid,a polynucleotide, RNA, single stranded DNA, double stranded DNA, anoligonucleotide, a peptide nucleic acid (PNA), a polypeptide, a protein,an antibody, a receptor, and a ligand.
 56. The method of claim 55,further comprising after step (a) reacting the activated slide surfacewith a multifunctional linker reagent comprising at least two reactivegroups capable of reacting with the silane to immobilize themultifunctional linker reagent on the surface, wherein the activatedsurface comprises the multifunctional linker reagent capable of reactingwith the target molecule to immobilize the target molecule on thesurface.
 57. The method of claim 55, wherein the target molecule is anucleic acid, a polynucleotide, a RNA, a single stranded DNA, a doublestranded DNA, an oligonucleotide, or a peptide nucleic acid.
 58. Themethod of claim 56, wherein the target molecule is a nucleic acid, apolynucleotide, a RNA, a single stranded DNA, a double stranded DNA, anoligonucleotide, or a peptide nucleic acid, and the multifunctionallinker reagent reactive group is an isothiocyanate and the linkercomprises from 1 to 20 carbon atoms.
 59. The method of claim 58, whereinthe multifunctional linker reagent comprises a plurality of linkermonomers.
 60. The method of claim 55, wherein the target moleculecomprises is unmodified.
 61. The method of claim 56, wherein the targetmolecule is modified and comprises an amine.
 62. The method of claim 61,wherein the amine is a primary amine at the 5′ end of the targetmolecule.
 63. The method of claim 55, wherein the silane is3-aminoproyltriethoxysilane.
 64. The method of claim 56, wherein themultifunctional linker reagent is 1,4-phenylene-diisothiocyanate.
 65. Amethod of preparing a detectably labeled sDNA probe capable of forming adetectable complex with a target molecule immobilized on a microarraysurface, the method comprising: (a) isolating an amount of totalcellular RNA from a biological sample; (b) synthesizing a mixture ofdetectably labeled sDNA probes, wherein the synthesis of sDNA comprisessynthesizing first strand cDNA from the isolated RNA of step (a),synthesizing second strand cDNA using Klenow fragment of DNA polymeraseI and the first strand cDNA as templates synthesizing cRNA using thedouble stranded cDNA as template; and synthesizing sDNA using reversetranscriptase in the presence of detectably labeled deoxyribonucleotideusing the cRNA as a template; (c) isolating the labeled sDNA probes. 66.The method of claim 65, wherein the amount of total cellular RNAcomprises from 0.01 to 10 pg messenger RNA.
 67. The method of claim 66,wherein the amount of total cellular RNA is from 1-5 pg.
 68. The methodof claim 67, wherein the amount of total cellular RNA is from 0.5-2 pg.69. The method of claim 65, wherein the synthesizing of sDNA is also inthe presence of hexamer primers under conditions that cause the sDNAprobes to have an average length of 0.5-2 kb.
 70. A method of preparinga detectably labeled cDNA probe capable of forming a detectable complexwith a target molecule immobilized on a microarray surface, the methodcomprising: (a) isolating an amount of total cellular RNA from abiological sample; (b) synthesizing a mixture of detectably labeled cDNAprobes, wherein the synthesis of cDNA comprises synthesizing firststrand cDNA from the isolated RNA of step (a) in the presence ordetectably labeled deoxynucleotide; (c) isolating the labeled sDNAprobes.
 71. A method of preparing a detectably labeled sDNA probecapable of forming a detectable complex with a target moleculeimmobilized on a microarray surface, the method comprising: (a)isolating an amount of total cellular RNA from a biological sample; (b)synthesizing a mixture of detectably labeled sDNA probes, wherein thesynthesis of sDNA comprises synthesizing a biotin-attached first strandcDNA from the isolated RNA of step (a); synthesizing second strand DNA(sDNA) using Klenow fragment of DNA polymerase I and the first strandcDNA as template in the presence of detectably labeled deoxynucleotides:(c) contacting the biotin-attached first strand cDNA with streptavidinand removing the biotin-first strand cDNA/streptavidin complex from thelabeled sDNA; and (c) isolating the labeled sDNA probes.
 72. A method ofpreparing a detectably labeled cRNA probe capable of forming adetectable complex with a target molecule immobilized on a microarraysurface, the method comprising: (a) isolating an amount of totalcellular RNA from a biological sample; (b) synthesizing a mixture ofdetectably labeled cRNA probes, wherein the synthesis of cRNA comprisessynthesizing first strand cDNA from the isolated RNA of step (a),synthesizing second strand cDNA using Klenow fragment of DNA polymeraseI and the first strand cDNA as template, synthesizing cRNA using thedouble stranded cDNA as template in the presence of delectably labeledribonucleotides; and (c) isolating the labeled sDNA probes.
 73. Themethod of claim 72, further comprising after step (c) degrading the cRNAprobe with RNase under conditions such that the average length of thecRNA probe is adjusted to be from 0.5 kb to 3 kb.
 74. The method ofclaim 71, wherein the step of synthesizing second strand DNA is in thepresence of hexamer primers under conditions such that the averagelength of the labeled sDNA probe is from approximately 0.5 kb toapproximately 2 kb.
 75. The method of claim 70, further comprising afterstep (b) decreasing the average length of the labeled cDNA probes to befrom 0.5 kb to 2 kb.
 76. The method of claim 75, wherein the decreasingis by limited DNase digestion.
 77. The method of claim 65, wherein thebiological sample is selected from the group consisting of a cell, atissue sample, a body fluid sample, and a mixture of syntheticoligonucleotides.
 78. The method of claim 65, wherein the amount oftotal cellular RNA is from 0.5 pg to and including 10 mg.
 79. The methodof claim 78, wherein the amount of total cellular RNA is from 1 pg toand including 10 μg.
 80. The method of claim 79, wherein the amount oftotal cellular RNA is from 1 pg to and including 100 ng.
 81. The methodof claim 80, wherein the amount of total cellular RNA is from 1 pg toand including 10 ng.
 82. The method of claim 65, wherein the detectablylabeled deoxynucleotide is labeled dUTP and the synthesizing in thepresence of labeled dUTP is in the absence of unlabeled dTTP.
 83. Themethod of claim 65, wherein the detectable label is a fluorochromophore.84. A method of analyzing a target molecule attached to a microarray,the method comprising: (a) providing a microarray according to claim 1;(b) contacting the attached target molecule with an agent capable offorming a detectable complex with the target molecule under conditionsthat allow formation of a detectable complex; (c) detecting formation ofa detectable complex; (d) determining the amount of a detectable complexformed.
 85. The method of claim 84, wherein the agent capable of forminga detectable complex comprises: a control mixture of sDNA probescomprising a first detectable label, wherein the probes are preparedfrom total cellular RNA isolated from a control sample, and a testmixture of sDNA probes comprising a second detectable label, wherein theprobes are prepared from total cellular RNA isolated from a test sample,wherein the first and second detectable labels are distinguishable,wherein the method further comprises: (1) pooling the control sDNAprobes and the test sDNA probes; (2) performing steps (a)-(d) of claim84; and (3) comparing the amount of detectable complex formed betweenthe target molecule and the control probes relative to the amount ofcomplex formed between the target molecule and the test probes.
 86. Themethod of claim 84, wherein the label is optically detectable.
 87. Themethod of claim 86, wherein the label is fluorescent.
 88. The method ofclaim 84, wherein the contacting of step (b) occurs in the absence ofdetergent.
 89. The method of claim 88, wherein the contacting of step(b) occurs in the presence of formamide and a one or more ofdimethylsulfoxide (DMSO), tetramethylammonium chloride (TMACl), andtetraethylammonium chloride (TEACl).
 90. The method of claim 89, whereinthe contacting of step (b) occurs in the presence of formamide, DMSO andTMACl or TEACl, wherein the sum of the proportions of formamide and DMSOdoes not exceed 50%.
 91. The method of claim 90, wherein the sum of theproportions of formamide and DMSO does not exceed 25%.
 92. The method ofclaim 88, further comprising a wash step subsequent to the contactingstep wherein the wash solution comprises detergent.
 93. A method ofhybridizing a detectable polynucleotide probe to a target polynucleotideon a support surface, the method comprising: (a) contacting the probewith denatured target polynucleotide on the support surface in ahybridization solution comprising DMSO or formamide or both, and in theabsence of detergent; and (b) detecting formation of a complex betweenthe target polynucleotide and the detectably labeled polynucleotideprobe.
 94. The method of claim 93, wherein the sum of the proportions ofDMSO and formamide does not exceed 50%, and wherein the hybridizationsolution further comprises TMACl or TMECl or both.
 95. The method ofclaim 94, wherein the sum of the proportions of DMSO and formamide doesnot exceed 25%, and wherein the hybridization solution further comprisesTMACl or TMECl or both.
 96. The method of claim 85, wherein the controlsample comprises cells removed from a cell source by laser capturemicrodissection, wherein the cell source is selected from the groupconsisting of untreated tissue, frozen tissue, paraffin-embedded tissue,stained tissue, and cell culture.
 97. The method of claim 85, whereinthe test sample comprises cells removed from a cell source by lasercapture microdissection, wherein the cell source is selected from thegroup consisting of untreated tissue, frozen tissue, paraffin-embeddedtissue, stained tissue, and cell culture.
 98. The method of claim 85,wherein the test sample and control sample differ according to one ormore of developmental state, disease state, pre-disease state, celltype, sample source, and experimental treatment conditions.
 99. Themethod of claim 85, wherein the target molecule is a polynucleotide andthe nucleic acid isolated from the test sample and the control sample isRNA, and wherein the comparing of step (c) provides a measure of targetpolynucleotide expression in the test sample relative to targetpolynucleotide expression in the control sample.
 100. The method ofclaim 99, wherein the relative measure of target polynucleotideexpression indicates a disease state in the test tissue sample.
 101. Themethod of claim 100, wherein the disease state is selected from thegroup consisting of tumor, cardiovasular disease, inflammatory disease,endocrine disease.
 102. The method of claim 100, wherein the relativemeasure of target polynucleotide expression indicates a pre-diseasestate in the test tissue sample.
 103. The method of claim 84, whereinthe target molecule is a polynucleotide and the nucleic acid isolatedfrom the test sample and the control sample is DNA, and wherein thecomparing of step (c) provides a measure of number of copies of thetarget polynucleotide in cells of the test sample relative to targetpolynucleotide copies in the control sample.
 104. The method of claim103, wherein the relative measure of the number of copies of targetpolynucleotide indicates a disease state or a pre-disease state in thetest tissue sample.